Cationic neurotoxins

ABSTRACT

The present invention provides engineered clostridial toxins comprising at least one amino acid modification, wherein said at least one amino acid modification increases the isoelectric point (pi) of the engineered clostridial toxin to a value that is at least 0.2 pi units higher than the pi of an otherwise identical clostridial toxin lacking said at least one amino acid modification, and wherein said at least one amino acid modification is not located in the clostridial toxin binding domain (Hc domain). Also provided are medical uses of the engineered clostridial toxins.

The present invention relates to engineered clostridial toxins comprising at least one amino acid modification, and the use of such engineered clostridial toxins in medicine and therapy.

Bacteria in the genus Clostridia produce highly potent and specific protein toxins, which can poison neurons and other cells to which they are delivered. Examples of such clostridial toxins include the neurotoxins produced by C. tetani (TeNT) and by C. botulinum (BoNT) serotypes A-G, as well as those produced by C. baratii and C. butyricum.

Among the clostridial toxins are some of the most potent toxins known. By way of example, botulinum neurotoxins have median lethal dose (LD₅ ₀ ) values for mice ranging from 0.5 to 5 ng/kg, depending on the serotype. Both tetanus and botulinum toxins act by inhibiting the function of affected neurons, specifically the release of neurotransmitters. While botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, tetanus toxin acts in the central nervous system.

In nature, clostridial toxins are synthesised as a single-chain polypeptide that is modified post-translationally by a proteolytic cleavage event to form two polypeptide chains joined together by a disulphide bond. Cleavage occurs at a specific cleavage site, often referred to as the activation site, that is located between the cysteine residues that provide the inter-chain disulphide bond. It is this di-chain form that is the active form of the toxin. The two chains are termed the heavy chain (H-chain), which has a molecular mass of approximately 100 kDa, and the light chain (L-chain), which has a molecular mass of approximately 50 kDa. The H-chain comprises an N-terminal translocation component (H_(N) domain) and a C-terminal targeting component (He domain). The cleavage site is located between the L-chain and the translocation domain components. Following binding of the ¾ domain to its target neuron and internalisation of the bound toxin into the cell via an endosome, the H_(N) domain translocates the L-chain across the endosomal membrane and into the cytosol, and the L-chain provides a protease function (also known as a non-cytotoxic protease).

Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g. SNAP-25, VAMP, or Syntaxin)—see Gerald K (2002) “Cell and Molecular Biology” (4th edition) John Wiley & Sons, Inc. The acronym SNARE derives from the term Soluble NSF Attachment Receptor, where NSF means N-ethylmaleimide-Sensitive Factor. SNARE proteins are integral to intracellular vesicle fusion, and thus to secretion of molecules via vesicle transport from a cell. The protease function is a zinc-dependent endopeptidase activity and exhibits a high substrate specificity for SNARE proteins. Accordingly, once delivered to a desired target cell, the non-cytotoxic protease is capable of inhibiting cellular secretion from the target cell. The L-chain proteases of clostridial toxins are non-cytotoxic proteases that cleave SNARE proteins.

In view of the ubiquitous nature of SNARE proteins, clostridial toxins such as botulinum toxin have been successfully employed in a wide range of therapies.

By way of example, we refer to William J. Lipham, Cosmetic and Clinical Applications of Botulinum Toxin (Slack, Inc., 2004), which describes the use of clostridial toxins, such as botulinum neurotoxins (BoNTs), BoNT/A, BoNT/B, BoNT/Ci, BoNT/D, BoNT/E, BoNT/F and BoNT/G, and tetanus neurotoxin (TeNT), to inhibit neuronal transmission in a number of therapeutic and cosmetic or aesthetic applications—for example, marketed botulinum toxin products are currently approved as therapeutics for indications including focal spasticity, upper limb spasticity, lower limb spasticity, cervical dystonia, blepharospasm, hemifacial spasm, hyperhidrosis of the axillae, chronic migraine, neurogenic detrusor overactivity, glabellar lines, and severe lateral canthal lines. In addition, clostridial toxin therapies are described for treating neuromuscular disorders (see U.S. Pat. No. 6,872,397); for treating uterine disorders (see US 2004/0175399); for treating ulcers and gastroesophageal reflux disease (see US 2004/0086531); for treating dystonia (see U.S. Pat. No. 6,319,505); for treating eye disorders (see US 2004/0234532); for treating blepharospasm (see US 2004/0151740); for treating strabismus (see US 2004/0126396); for treating pain (see U.S. Pat. Nos. 6,869,610, 6,641,820, 6,464,986, and 6,113,915); for treating fibromyalgia (see U.S. Pat. No. 6,623,742, US 2004/0062776); for treating lower back pain (see US 2004/0037852); for treating muscle injuries (see U.S. Pat. No. 6,423,319); for treating sinus headache (see U.S. Pat. No. 6,838,434); for treating tension headache (see U.S. Pat. No. 6,776,992); for treating headache (see U.S. Pat. No. 6,458,365); for reduction of migraine headache pain (see U.S. Pat. No. 5,714,469); for treating cardiovascular diseases (see U.S. Pat. No. 6,767,544); for treating neurological disorders such as Parkinson's disease (see U.S. Pat. Nos. 6,620,415, 6,306,403); for treating neuropsychiatric disorders (see US 2004/0180061, US 2003/021 1121); for treating endocrine disorders (see U.S. Pat. No. 6,827,931); for treating thyroid disorders (see U.S. Pat. No. 6,740,321); for treating cholinergic influenced sweat gland disorders (see U.S. Pat. No. 6,683,049); for treating diabetes (see U.S. Pat. Nos. 6,337,075, 6,416,765); for treating a pancreatic disorder (see U.S. Pat. Nos. 6,261,572, 6,143,306); for treating cancers such as bone tumors (see U.S. Pat. Nos. 6,565,870, 6,368,605, 6,139,845, US 2005/0031648); for treating otic disorders (see U.S. Pat. Nos. 6,358,926, 6,265,379); for treating autonomic disorders such as gastrointestinal muscle disorders and other smooth muscle dysfunction (see U.S. Pat. No. 5,437,291); for treatment of skin lesions associated with cutaneous cell-proliferative disorders (see U.S. Pat. No. 5,670,484); for management of neurogenic inflammatory disorders (see U.S. Pat. No. 6,063,768); for reducing hair loss and stimulating hair growth (see U.S. Pat. No. 6,299,893); for treating downturned mouth (see U.S. Pat. No. 6,358,917); for reducing appetite (see US 2004/40253274); for dental therapies and procedures (see US 2004/0115139); for treating neuromuscular disorders and conditions (see US 2002/0010138); for treating various disorders and conditions and associated pain (see US 2004/0013692); for treating conditions resulting from mucus hypersecretion such as asthma and COPD (see WO 00/10598); and for treating non-neuronal conditions such as inflammation, endocrine conditions, exocrine conditions, immunological conditions, cardiovascular conditions, bone conditions (see WO 01/21213). All of the above publications are hereby incorporated by reference in their entirety.

The use of non-cytotoxic proteases such as clostridial toxins (e.g. BoNTs and TeNT) in therapeutic and cosmetic treatments of humans and other mammals is anticipated to expand to an ever-widening range of diseases and ailments that can benefit from the properties of these toxins.

To avoid systemic neurological effects, many clostridial toxin therapies utilise direct administration of the clostridial toxin therapeutic to a given target site (such as a target tissue). A problem when administering clostridial toxin-based therapeutics in this fashion is the spread of toxin away from the administration site and into surrounding tissue or systemic circulation. The diffusion of toxin away from the target tissue is believed to be responsible for undesirable side effects that in extreme cases may be life threatening. This can be a particular concern when using clostridial toxin therapeutics (such as BoNT therapeutics) at high doses, concentrations and injection volumes. Adverse effects associated with this problem that have been reported for commercial BoNT/A therapeutics include asthenia, generalised muscle weakness, diplopia, ptosis, dysphagia, dysphonia, dysarthria, urinary incontinence, and breathing difficulties. Swallowing and breathing difficulties can be life threatening and there have been reported deaths related to the spread of toxin effects.

There is therefore a need in the art for clostridial toxins which have properties of increased tissue retention at the site of administration, and which accordingly exhibit a reduction in diffusion away from the administration site, as compared to known clostridial toxins.

The present invention solves the above problem by providing engineered clostridial toxins, as specified in the claims.

In one aspect, the invention provides an engineered clostridial toxin comprising at least one (for example, at least one, two or three) amino acid modification, wherein said at least one amino acid modification increases the isoelectric point (pi) of the engineered clostridial toxin to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical clostridial toxin lacking said at least one amino acid modification, and wherein said at least one amino acid modification is not located in the clostridial toxin binding domain (¾ domain).

In one embodiment, “not located in the clostridial toxin binding domain (¾) domain” means that said at least one amino acid modification is located in the clostridial toxin H_(N) domain or in the clostridial toxin light chain.

In one embodiment, the invention provides an engineered clostridial toxin comprising at least one (for example, at least one, two or three) amino acid modification, wherein said at least one amino acid modification increases the isoelectric point (pi) of the engineered clostridial toxin to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical clostridial toxin lacking said at least one amino acid modification, and wherein said at least one amino acid modification is located in the clostridial toxin translocation domain (H_(N) domain).

In another embodiment, the invention provides an engineered clostridial toxin comprising at least one (for example, at least one, two or three) amino acid modification, wherein said at least one amino acid modification increases the isoelectric point (pi) of the engineered clostridial toxin to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical clostridial toxin lacking said at least one amino acid modification, and wherein said at least one amino acid modification is located in the clostridial toxin light chain.

In one embodiment, wherein said at least one amino acid modification is located in the clostridial toxin light chain, said at least one amino acid modification does not introduce into the clostridial toxin light chain an E3 ligase recognition motif. Thus, in one embodiment, the light chain of an engineered clostridial toxin of the invention does not comprise an E3 ligase recognition motif.

As used above, the term “E3 ligase recognition motif refers to a modification of the light chain that results in accelerated degradation of the neurotoxin polypeptide by endogenous proteasome degradation pathways present in a subject to which the neurotoxin has been applied. An “E3 ligase recognition” motif is a structural motif that allows recognition of the motif and binding to the motif by an E3 ligase (also known as an E3 ubiquitin ligase; thus, an “E3 ligase recognition motif may also be referred to as an “E3 ubiquitin ligase recognition motif). E3 ligase recognition motifs will be familiar to a person skilled in the art.

Examples of E3 ligase recognition motifs include the following sequences (wherein “X” may represent any of the naturally occurring amino acids):

E3 ubiquitin Recognition motif ligase (consensus) VBCCu12 ALAPYIP (SEQ ID NO: 9) MDM2 XXFXXXWXXLXX (SEQ ID NO: 10) MNM2 RFMDYWEGL (SEQ ID NO: 11) FXXXLWXXL (SEQ ID NO: 12) Smurf2 ELESPPPPYSRYPM (SEQ ID NO: 13) RN181 KVGFFKR (SEQ ID NO: 14) E3alpha LLVRGRTLVV (SEQ ID NO: 15) SCF DRHDSGLDSM (SEQ ID NO: 16) Siah PXAXVXP (SEQ ID NO: 17) Itch PPXYXXM (SEQ ID NO:18) Nedd4-2 PPXY (SEQ ID NO: 19)

Further examples of E3 ligase recognition motifs include:

(SEQ ID NO: 20) ETFSDLWKLLPE, (SEQ ID NO: 21) TSFAEYWNLLSP, (SEQ ID NO: 22) LTFEHYWAQLTS, (SEQ ID NO: 23) LTFEHWWAQLTS, (SEQ ID NO: 24) LTFEHSWAQLTS, (SEQ ID NO: 25) ETFEHNWAQLTS, (SEQ ID NO: 26) LTFEHNWAQLTS, (SEQ ID NO: 27) LTFEHWWASLTS, (SEQ ID NO: 28) LTFEHWWSSLTS, (SEQ ID NO: 29) LTFTHWWAQLTS, (SEQ ID NO: 30) ETFEHWWAQLTS, (SEQ ID NO: 31) LTFEHWWSQLTS, (SEQ ID NO: 32) LTFEHWWAQLLS, (SEQ ID NO: 33) ETFEHWWSQLLS, (SEQ ID NO: 34) RFMDYWEGL, (SEQ ID NO: 35) MPRFMDYWEGLN, (SEQ ID NO: 36) SQETFSDLWKLLPEN, and (SEQ ID NO: 37) LTFEHNWAQLEN.

In one embodiment, wherein said at least one amino acid modification is located in the clostridial toxin light chain, said at least one amino acid modification does not introduce into the clostridial toxin light chain an MDM2 E3 ligase recognition motif. Thus, in one embodiment, the light chain of an engineered clostridial toxin of the invention does not comprise an MDM2 E3 ligase recognition motif.

In one embodiment, wherein said at least one amino acid modification is located in the clostridial toxin light chain, the engineered clostridial toxin does not comprise an amino acid modification at an N-terminal proline.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E, and wherein said at least one amino acid modification is located in the clostridial toxin light chain, said engineered BoNT/E does not comprise a substitution with lysine at any one of the following amino acid positions: Q53, N72, N378, N379, R394, T400.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E, and wherein said at least one amino acid modification is located in the clostridial toxin light chain, said engineered BoNT/E does not comprise a substitution with lysine at any one of the following amino acid positions: Q53, N72, N378, N379, T400.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E, and wherein said at least one amino acid modification is located in the clostridial toxin light chain, said engineered BoNT/E does not comprise a substitution with lysine at any three of the following amino acid positions: Q53, N72, N378, N379, R394, T400.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E, and wherein said at least one amino acid modification is located in the clostridial toxin light chain, said engineered BoNT/E does not comprise a substitution with lysine at any three of the following amino acid positions: Q53, N72, N378, N379, T400.

In one embodiment, optionally wherein the at least one amino acid modification is located in the clostridial toxin light chain, the engineered clostridial toxin is not a BoNT/E.

The engineered clostridial toxins of the invention do not comprise any amino acid modifications located in the clostridial toxin He domain. Thus, in an engineered clostridial toxin of the invention, said at least one amino acid modification is not located in the clostridial toxin ¾ domain.

In one embodiment, wherein said at least one amino acid modification is located in the clostridial toxin light chain, said at least one amino acid modification does not comprise the substitution of an amino acid residue with a lysine residue.

In one embodiment, wherein the engineered clostridial toxin is an engineered clostridial toxin as described above, said at least one amino acid modification comprises substitution of an acidic amino acid residue or an uncharged amino acid residue with a lysine or arginine residue.

In one embodiment, wherein the engineered clostridial toxin is an engineered clostridial toxin as described above, said at least one amino acid modification comprises substitution of an acidic amino acid residue or an uncharged amino acid residue with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is an engineered clostridial toxin as described above, said at least one amino acid modification increases the pi of the engineered clostridial toxin to a value that is at least 0.4 pi units higher than the pi of an otherwise identical clostridial toxin lacking said at least one amino acid modification. In one embodiment, said at least one amino acid modification increases the pi of the engineered clostridial toxin to a value that is at least 0.5 pi units higher than the pi of an otherwise identical clostridial toxin lacking said at least one amino acid modification. In one embodiment, said at least one amino acid modification increases the pi of the engineered clostridial toxin to a value that is at least 0.6 pi units higher than the pi of an otherwise identical clostridial toxin lacking said at least one amino acid modification. In one embodiment, said at least one amino acid modification increases the pi of the engineered clostridial toxin to a value that is at least 0.8 pi units higher than the pi of an otherwise identical clostridial toxin lacking said at least one amino acid modification. In one embodiment, said at least one amino acid modification increases the pi of the engineered clostridial toxin to a value that is at least 1 pi unit higher than the pi of an otherwise identical clostridial toxin lacking said at least one amino acid modification.

In certain embodiments, the engineered clostridial toxin comprises at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85 or 90 amino acid modifications.

In certain embodiments, said at least one amino acid modification increases the pi of the engineered clostridial toxin to a value that is at least 2, 3, 4 or 5 pi units higher than the pi of an otherwise identical clostridial toxin lacking said at least one amino acid modification.

In certain embodiments, the engineered clostridial toxin comprises at least 3 amino acid modifications, and said at least 3 amino acid modifications increase the pi of the engineered clostridial toxin to a value that is at least 0.2 pi units higher than the pi of an otherwise identical clostridial toxin lacking said at least 3 amino acid modifications.

In certain embodiments, the engineered clostridial toxin comprises at least 5 amino acid modifications, and said at least 5 amino acid modifications increase the pi of the engineered clostridial toxin to a value that is at least 0.5 pi units higher than the pi of an otherwise identical clostridial toxin lacking said at least 5 amino acid modifications.

The present inventors have found that by increasing the pi of a clostridial toxin, for example, by at least 0.2 pi units, or 0.5 pi units, or one pi unit (through the introduction into the clostridial toxin protein of at least one amino acid modification), the resultant engineered clostridial toxin advantageously demonstrates properties of increased tissue retention and reduced diffusion away from sites of administration, while retaining abilities of target cell binding, translocation, and cleavage of target SNARE protein(s). Thus, the spread of clostridial toxin from the site of administration is significantly reduced, as compared to an otherwise identical clostridial toxin lacking said at least one amino acid modification.

The engineered clostridial toxins of the invention are suitable for use in any of the therapies described above, and advantageously may demonstrate a reduction in, or absence of, side effects compared to the use of known clostridial toxin therapeutics.

The increased tissue retention properties of the engineered clostridial toxins of the invention also provide increased potency and/or duration of action, and can allow for reduced dosages to be used compared to known clostridial toxin therapeutics (or increased dosages without any additional adverse effects), thus providing further advantages.

Thus, in one embodiment, an engineered clostridial toxin of the invention has increased potency, increased tissue retention, and/or increased duration of action, as compared to the corresponding unmodified clostridial toxin.

As discussed below in more detail, the increase in pi provided by the at least one amino acid modification means that an engineered clostridial toxin of the invention has, at a given pH, a net charge that is more positive than the net charge on an otherwise identical clostridial toxin lacking said at least one amino acid modification. Without wishing to be bound by any one theory, the present inventors believe that this increased positive charge allows the engineered clostridial toxins of the present invention to display longer tissue retention times at the site of administration due to favourable electrostatic interactions between the engineered clostridial toxin and anionic extracellular components (such as cell membranes and heparin sulphate proteoglycans) at the site of administration. These improved electrostatic interactions serve to reduce the diffusion of the engineered clostridial toxin away from the site of administration, thus improving tissue retention.

By way of example, the improved tissue retention properties of an engineered clostridial toxin of the invention may allow for (i) higher doses into individual muscles, such as the sternocleidomastoid, without spreading into nearby muscles in the neck to cause difficult swallowing, and (ii) higher total doses (to all muscles) in a single treatment, without spreading into the circulation and causing systemic effects such as difficult breathing. Advantages to patients may include more effective treatment of large muscles such as the sternocleidomastoid muscle, increased opportunity to inject several different muscles during each treatment, and possible longer duration of effective treatment (longer before re-treatment is necessary) because of higher dosing.

In one embodiment, an engineered clostridial toxin of the invention has, in use, a positive net charge (for example, when the engineered clostridial toxin, in use, is located at a desired administration site in a tissue).

The isoelectric point (pi) is a specific property of a given protein. As is well known in the art, proteins are made from a specific sequence of amino acids (also referred to when in a protein as amino acid residues). Each amino acid of the standard set of twenty has a different side chain (or R group), meaning that each amino acid residue in a protein displays different chemical properties such as charge and hydrophobicity. These properties may be influenced by the surrounding chemical environment, such as the temperature and pH. The overall chemical characteristics of a protein will depend on the sum of these various factors.

Certain amino acid residues (detailed below) possess ionisable side chains that may display an electric charge depending on the surrounding pH. Whether such a side chain is charged or not at a given pH depends on the pKa of the relevant ionisable moiety, wherein pKa is the negative logarithm of the acid dissociation constant (Ka) for a specified proton from a conjugate base.

For example, acidic residues such as aspartic acid and glutamic acid have side chain carboxylic acid groups with pKa values of approximately 4.1 (precise pKa values may depend on temperature, ionic strength and the microenvironment of the ionisable group). Thus, these side chains exhibit a negative charge at a pH of 7.4 (often referred to as “physiological pH”). At low pH values, these side chains will become protonated and lose their charge.

Conversely, basic residues such as lysine and arginine have nitrogen-containing side chain groups with pKa values of approximately 10-12. These side chains therefore exhibit a positive charge at a pH of 7.4. These side chains will become de-protonated and lose their charge at high pH values.

The overall (net) charge of a protein molecule therefore depends on the number of acidic and basic residues present in the protein (and their degree of surface exposure) and on the surrounding pH. Changing the surrounding pH changes the overall charge on the protein. Accordingly, for every protein there is a given pH at which the number of positive and negative charges is equal and the protein displays no overall net charge. This point is known as the isoelectric point (pi). The isoelectric point is a standard concept in protein biochemistry with which the skilled person would be familiar.

The isoelectric point (pi) is therefore defined as the pH value at which a protein displays a net charge of zero. An increase in pi means that a higher pH value is required for the protein to display a net charge of zero. Thus, an increase in pi represents an increase in the net positive charge of a protein at a given pH.

Conversely, a decrease in pi means that a lower pH value is required for the protein to display a net charge of zero. Thus, a decrease in pi represents a decrease in the net positive charge of a protein at a given pH.

Methods of determining the pi of a protein are known in the art and would be familiar to a skilled person. By way of example, the pi of a protein can be calculated from the average pKa values of each amino acid present in the protein (“calculated pi”). Such calculations can be performed using computer programs known in the art; preferred example computer programs for calculating pi values include Protein Calculator from the Scripps Research Institute and Compute pI/MW Tool from ExPASy. Comparisons of pi values between different molecules should be made using the same calculation technique/program.

Where appropriate, the calculated pi of a protein can be confirmed experimentally using the technique of isoelectric focusing (“observed pi”). This technique uses electrophoresis to separate proteins according to their pi. Isoelectric focusing is typically performed using a gel that has an immobilised pH gradient. When an electric field is applied, the protein migrates through the pH gradient until it reaches the pH at which it has zero net charge, this point being the pi of the protein. Results provided by isoelectric focusing are typically relatively low-resolution in nature, and thus the present inventors believe that results provided by calculated pi (as described above) are more appropriate to use.

Throughout the present specification, “pi” means “calculated pi” unless otherwise stated.

The pi of a protein may be increased or decreased by altering the number of basic and/or acidic groups displayed on its surface. This can be achieved by modifying one or more amino acids of the protein. For example, an increase in pi may be provided by reducing the number of acidic residues, or by increasing the number of basic residues. Such amino acid modifications are discussed in more detail below.

Native (unmodified) clostridial toxins have a pi of approximately 5-6. Thus, at a pH of 7.4, native botulinum toxins possess a negative net charge. By way of example, the pi of BoNT/A is 6.4, and a BoNT/A molecule has a net charge at pH 7.4 of −8. These pi values are calculated as described above.

TABLE 1 CLOSTRIDIAL TOXIN pI BoNT/A 6.4 BoNT/B 5.3 BoNT/C₁ 5.5 BoNT/D 5.5 BoNT/E 6.0 BoNT/F 5.6 BoNT/G 5.2 TeNT 5.8

As described above, in one embodiment, an engineered clostridial toxin of the present invention comprises at least one amino acid modification, wherein said at least one amino acid modification increases the isoelectric point (pi) of the engineered clostridial toxin to a value that is at least 0.2 pi units higher than the pi of an otherwise identical clostridial toxin lacking said at least one amino acid modification.

Thus, in the context of the present invention, an increase in pi of 0.2 units in the context of an engineered BoNT/A clostridial toxin would be an increase in pi from 6.4 to 6.6.

As described above, in one embodiment, an engineered clostridial toxin of the present invention comprises at least one amino acid modification, wherein said at least one amino acid modification increases the isoelectric point (pi) of the engineered clostridial toxin to a value that is at least one pi unit higher than the pi of an otherwise identical clostridial toxin lacking said at least one amino acid modification.

Thus, in the context of the present invention, an increase in pi of 1 unit in the context of an engineered BoNT/A clostridial toxin would be an increase in pi from 6.4 to 7.4.

In one embodiment, the engineered clostridial toxin has a pi of at least 5.5.

In one embodiment, the engineered clostridial toxin has a pi of at least 6 (for example, at least 6, at least 7, at least 8, or at least 9).

In one embodiment, the engineered clostridial toxin has a pi of at least 6.5.

In one embodiment, the engineered clostridial toxin has a pi of at least 7.

In one embodiment, the engineered clostridial toxin has a pi of between 6.5 and 9.5 (for example a pi of between 6.5 and 7.5).

As discussed above, the engineered clostridial toxins of the present invention have increased tissue retention properties that also provide increased potency and/or duration of action, and can allow for reduced dosages to be used compared to known clostridial toxin therapeutics (or increased dosages without any additional effects). One way in which these advantageous properties (which represent an increase in the therapeutic index) may be defined is in terms of the Safety Ratio of the engineered clostridial toxin. In this regard, undesired effects of a clostridial toxin (caused by diffusion of the toxin away from the site of administration) can be assessed experimentally by measuring percentage bodyweight loss in a relevant animal model (e.g. a mouse, where loss of bodyweight is detected within seven days of administration). Conversely, desired on-target effects of a clostridial toxin can be assessed experimentally by Digital Abduction Score (DAS) assay, a measurement of muscle paralysis. The DAS assay may be performed by injection of 20 μl of clostridial toxin, formulated in Gelatin Phosphate Buffer, into the mouse gastrocnemius/soleus complex, followed by assessment of Digital Abduction Score using the method of Aoki (Aoki K R, Toxicon 39: 1815-1820; 2001). In the DAS assay, mice are suspended briefly by the tail in order to elicit a characteristic startle response in which the mouse extends its hind limbs and abducts its hind digits. Following clostridial toxin injection, the varying degrees of digit abduction are scored on a five-point scale (0=normal to 4=maximal reduction in digit abduction and leg extension).

The Safety Ratio of a clostridial toxin may then be expressed as the ratio between the amount of toxin required for a 10% drop in a bodyweight (measured at peak effect within the first seven days after dosing in a mouse) and the amount of toxin required for a DAS score of 2. High Safety Ratio scores are therefore desired, and indicate a toxin that is able to effectively paralyse a target muscle with little undesired off-target effects. An engineered toxin of the present invention has a Safety Ratio that is higher than the Safety Ratio of an equivalent unmodified (native) botulinum toxin.

Thus, in one embodiment, an engineered clostridial toxin of the present invention has a Safety Ratio of at least 8 (for example, at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50), wherein Safety Ratio is calculated as: dose of toxin required for −10% bodyweight change (pg/mouse) divided by DAS ED₅ ₀ (pg/mouse) [ED₅ ₀ =dose required to produce a DAS score of 2].

In one embodiment, an engineered clostridial toxin of the present invention has a Safety Ratio of at least 10. In one embodiment, an engineered clostridial toxin of the present invention has a Safety Ratio of at least 15.

An engineered clostridial toxin of the present invention comprises at least one amino acid modification. Said at least one amino acid modification increases the pi of the clostridial toxin, as discussed above. In the context of the present invention, an amino acid modification is a modification of the amino acid sequence of a clostridial toxin.

Such a modification may be effected by replacing one amino acid in the sequence with another (i.e. a substitution), by inserting a new amino acid into the sequence, or by deleting an amino acid of the sequence. Amino acids incorporated into an amino acid sequence in a protein are also referred to as amino acid residues.

The 20 standard amino acids found in proteins are as follows:

TABLE 2 AMINO ACID SIDE CHAIN Aspartic acid Asp D Charged (acidic) Glutamic acid Glu E Charged (acidic) Arginine Arg R Charged (basic) Lysine Lys K Charged (basic) Histidine His H Uncharged (polar) Asparagine Asn N Uncharged (polar) Glutamine Gln Q Uncharged (polar) Serine Ser S Uncharged (polar) Threonine Thr T Uncharged (polar) Tyrosine Tyr Y Uncharged (polar) Methionine Met M Uncharged (polar) Tryptophan Trp W Uncharged (polar) Cysteine Cys C Uncharged (polar) Alanine Ala A Uncharged (hydrophobic) Glycine Gly G Uncharged (hydrophobic) Valine Val V Uncharged (hydrophobic) Leucine Leu L Uncharged (hydrophobic) Isoleucine Ile I Uncharged (hydrophobic) Proline Pro P Uncharged (hydrophobic) Phenylalanine Phe F Uncharged (hydrophobic)

The following amino acids are considered charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive), and lysine (positive).

At a pH of 7.4, the side chains of aspartic acid (pKa 3.1) and glutamic acid (pKa 4.1) have a negative charge, while the side chains of arginine (pKa 12.5) and lysine (pKa 10.8) have a positive charge. Aspartic acid and glutamic acid are referred to as acidic amino acid residues. Arginine and lysine are referred to as basic amino acid residues.

The following amino acids are considered uncharged, polar (meaning they can participate in hydrogen bonding) amino acids: asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, tryptophan.

The following amino acids are considered uncharged, hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine.

An increase in the pi of a clostridial toxin can be effected by introducing into the clostridial toxin one or more amino acid modifications that increases the ratio of positive to negative charges in the clostridial toxin.

In one embodiment, the at least one amino acid modification is selected from: an amino acid substitution, an amino acid insertion, and an amino acid deletion.

In an amino acid substitution, an amino acid residue that forms part of the clostridial toxin amino acid sequence is replaced with a different amino acid residue. The replacement amino acid residue may be one of the 20 standard amino acids, as described above.

Alternatively, the replacement amino acid in an amino acid substitution may be a non-standard amino acid (an amino acid that is not part of the standard set of 20 described above). By way of example, the replacement amino acid may be a basic non-standard amino acid, e.g. L-Ornithine, L-2-amino-3-guanidinopropionic acid, or D-isomers of Lysine, Arginine and Ornithine). Methods for introducing non-standard amino acids into proteins are known in the art, and include recombinant protein synthesis using E. coli auxotrophic expression hosts.

In an amino acid insertion, an additional amino acid residue (one that is not normally present) is incorporated into the clostridial toxin amino acid sequence, thus increasing the total number of amino acid residues in said sequence. In an amino acid deletion, an amino acid residue is removed from the clostridial toxin amino acid sequence, thus reducing the total number of amino acid residues in said sequence.

Methods for modifying proteins by substitution, insertion or deletion of amino acid residues are known in the art. By way of example, amino acid modifications may be introduced by modification of a DNA sequence encoding a clostridial toxin. This can be achieved using standard molecular cloning techniques, for example by site-directed mutagenesis where short strands of DNA (oligonucleotides) coding for the desired amino acid(s) are used to replace the original coding sequence using a polymerase enzyme, or by inserting/deleting parts of the gene with various enzymes (e.g., ligases and restriction endonucleases). Alternatively a modified gene sequence can be chemically synthesised.

In one embodiment, the at least one amino acid modification is selected from: substitution of an acidic amino acid residue with a basic amino acid residue; substitution of an acidic amino acid residue with an uncharged amino acid residue; substitution of an uncharged amino acid residue with a basic amino acid residue; insertion of a basic amino acid residue; and deletion of an acidic amino acid residue.

In a preferred embodiment, the at least one amino acid modification is a substitution, which advantageously maintains the same number of amino acid residues in the clostridial toxin. In one embodiment, the substitution is selected from: substitution of an acidic amino acid residue with a basic amino acid residue, substitution of an acidic amino acid residue with an uncharged amino acid residue, and substitution of an uncharged amino acid residue with a basic amino acid residue. In one embodiment, the basic amino acid residue is a lysine residue or an arginine residue. In one embodiment, the basic amino acid residue is a lysine residue. In one embodiment, the basic amino acid residue is an arginine residue. In one embodiment, wherein the substitution is a substitution of an acidic amino acid residue with an uncharged amino acid residue, the acidic amino acid residue is replaced with its corresponding uncharged amide amino acid residue (i.e. aspartic acid is replaced with asparagine, and glutamic acid is replaced with glutamine).

In another preferred embodiment, the at least one amino acid modification is a substitution of an acidic amino acid residue with a basic amino acid residue, or a substitution of an uncharged amino acid residue with a basic amino acid residue. In one embodiment, the basic amino acid residue is a lysine residue. In one embodiment, the basic amino acid residue is an arginine residue.

An engineered clostridial toxin of the invention may comprise more than one amino acid modification. Thus, in one embodiment, the engineered clostridial toxin (as described above) comprises between 1 and 90 amino acid modifications (for example, between 1 and 80, between 1 and 70, between 1 and 60, between 1 and 50, between 1 and 40, between 1 and 30, between 1 and 20, between 1 and 10, between 3 and 50, between 3 and 40, between 3 and 30, between 4 and 40, between 4 and 30, between 5 and 40, between 5 and 30, or between 10 and 25 amino acid modifications). In one embodiment, the engineered clostridial toxin (as described above) comprises between 1 and 20 amino acid modifications. In one embodiment, the engineered clostridial toxin (as described above) comprises between 1 and 10 amino acid modifications. In one embodiment, the engineered clostridial toxin (as described above) comprises between 2 and 20 amino acid modifications. In one embodiment, the engineered clostridial toxin (as described above) comprises between 2 and 15 amino acid modifications. In one embodiment, the engineered clostridial toxin (as described above) comprises between 2 and 10 amino acid modifications. In one embodiment, the engineered clostridial toxin (as described above) comprises between 3 and 20 amino acid modifications. In one embodiment, the engineered clostridial toxin (as described above) comprises between 3 and 15 amino acid modifications. In one embodiment, the engineered clostridial toxin (as described above) comprises between 3 and 10 amino acid modifications. In one embodiment, the engineered clostridial toxin (as described above) comprises between 4 and 40 amino acid modifications. In one embodiment, the engineered clostridial toxin comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid modifications. In one embodiment, the engineered clostridial toxin comprises at least 3 amino acid modifications (for example, at least 3 amino acid substitutions). In one embodiment, the engineered clostridial toxin comprises at least 4 amino acid modifications (for example, at least 4 amino acid substitutions). Each of said amino acid modifications is an amino acid modification as described above. Thus, each of said amino acid modifications contributes to the increase in pi of the engineered clostridial toxin (as compared to the pi of an otherwise identical clostridial toxin lacking said amino acid modifications).

Any clostridial toxin amino acid (i.e. amino acid residue) that is not located in the clostridial toxin binding domain (¾ domain) can be modified as described above, as long as the outcome of said modification is an increase in the clostridial toxin pi (as described above). However, the present inventors have identified subsets of clostridial toxin amino acids that are particularly suitable targets for modification.

Preferred target amino acids may possess certain qualities. By way of example, a preferred target amino acid may be: (i) a surface exposed amino acid; (ii) located outside of a clostridial toxin protein secondary structure; (iii) located in a clostridial toxin protein region that is non-essential for protein function; (iv) an amino acid whose identity is not conserved between clostridial toxin types, subtypes, or serotypes; (iv) an amino acid whose modification does not create a predicted ubiquitination site; or (v) any combination of the foregoing.

As described above, the engineered clostridial toxins of the invention feature one or more amino acid modifications located in either the clostridial toxin H_(N) translocation domain or the clostridial toxin light chain.

Examples of clostridial toxin light chain reference sequences include:

Botulinum type A neurotoxin: amino acid residues 1-448 Botulinum type B neurotoxin: amino acid residues 1-440 Botulinum type Ci neurotoxin: amino acid residues 1-441 Botulinum type D neurotoxin: amino acid residues 1-445 Botulinum type E neurotoxin: amino acid residues 1-422 Botulinum type F neurotoxin: amino acid residues 1-439 Botulinum type G neurotoxin: amino acid residues 1-441 Tetanus neurotoxin: amino acid residues 1-457

Examples of clostridial toxin H_(N) domain reference sequences include:

Botulinum type A neurotoxin: amino acid residues 449-871 Botulinum type B neurotoxin: amino acid residues 443-862 Botulinum type Ci neurotoxin: amino acid residues 450-866 Botulinum type D neurotoxin: amino acid residues 449-871 Botulinum type E neurotoxin: amino acid residues 455-845 Botulinum type F neurotoxin: amino acid residues 450-864 Botulinum type G neurotoxin: amino acid residues 449-871 Tetanus neurotoxin: amino acid residues 456-879

Examples of clostridial toxin ¾ domain reference sequences include:

Botulinum type A neurotoxin: amino acid residues 872-1278 Botulinum type B neurotoxin: amino acid residues 863-1291 Botulinum type Ci neurotoxin: amino acid residues 867-1291 Botulinum type D neurotoxin: amino acid residues 872-1276 Botulinum type E neurotoxin: amino acid residues 846-1252 Botulinum type F neurotoxin: amino acid residues 865-1278 Botulinum type G neurotoxin: amino acid residues 872-1297 Tetanus neurotoxin: amino acid residues 880-1315

The above-identified reference sequences should be considered a guide, as slight variations may occur according to sub-serotypes. By way of example, US 2007/0166332 (hereby incorporated by reference in its entirety) cites slightly different clostridial sequences:

Light chain: Botulinum type A neurotoxin: amino acid residues M1-K448 Botulinum type B neurotoxin: amino acid residues M1-K441 Botulinum type Ci neurotoxin: amino acid residues M1-K449 Botulinum type D neurotoxin: amino acid residues M1-R445 Botulinum type E neurotoxin: amino acid residues M1-R422 Botulinum type F neurotoxin: amino acid residues M1-K439 Botulinum type G neurotoxin: amino acid residues M1-K446 Tetanus neurotoxin: amino acid residues M1-A457 H_(N) domain: Botulinum type A neurotoxin: amino acid residues A449-K871 Botulinum type B neurotoxin: amino acid residues A442-S858 Botulinum type Ci neurotoxin: amino acid residues T450-N866 Botulinum type D neurotoxin: amino acid residues D446-N862 Botulinum type E neurotoxin: amino acid residues K423-K845 Botulinum type F neurotoxin: amino acid residues A440-K864 Botulinum type G neurotoxin: amino acid residues S447-S863 Tetanus neurotoxin: amino acid residues S458-V879 He domain: Botulinum type A neurotoxin: amino acid residues N872-L1296 Botulinum type B neurotoxin: amino acid residues E859-E1291 Botulinum type Ci neurotoxin: amino acid residues N867-E1291 Botulinum type D neurotoxin: amino acid residues S863-E1276 Botulinum type E neurotoxin: amino acid residues R846-K1252 Botulinum type F neurotoxin: amino acid residues K865-E1274 Botulinum type G neurotoxin: amino acid residues N864-E1297 Tetanus neurotoxin: amino acid residues I880-D1315

In one embodiment, wherein said at least one amino acid modification is located in the clostridial toxin translocation domain (H_(N) domain), said at least one amino acid modification is not located in the clostridial toxin belt region. The clostridial toxin belt region (as determined by visual inspection of structures and models) is defined as follows:

Botulinum type A neurotoxin: amino acid residues 492-545 Botulinum type B neurotoxin: amino acid residues 472-532 Botulinum type Ci neurotoxin: amino acid residues 494-543 Botulinum type D neurotoxin: amino acid residues 489-539 Botulinum type E neurotoxin: amino acid residues 466-515 Botulinum type F neurotoxin: amino acid residues 485-536 Botulinum type G neurotoxin: amino acid residues 489-538 Tetanus neurotoxin: amino acid residues 506-556

In one embodiment, the at least one amino acid modification (as described above) is a modification of a surface exposed amino acid residue. Surface exposed amino acid residues are those present on the exterior of a folded protein and so accessible to the surrounding solvent, in contrast to those amino acid residues that are located in the interior of a folded protein. The degree of surface exposure of an amino acid residue and thus its exposure to the surrounding solvent depends on its position within the folded protein, and also on the conformation adopted by the protein. Modification of an amino acid residue with a high degree of surface exposure may therefore have a greater effect on the protein's isoelectric point than modification of an amino acid residue with a low degree of surface exposure. Methods for determining the degree of surface exposure of an amino acid residue are known in the art. By way of example, the computer program ArealMol (part of the CCP4 suite of computer programs) can be used to calculate the degree of surface exposure of amino acid residues in a given protein. Surface exposed amino acid residues may also be identified by visual inspection of a protein crystal structure (such as provided by X-ray crystallography). In one embodiment, a surface exposed amino acid residue has a sum ArealMol value of at least 40.

In one embodiment, the at least one amino acid modification comprises modification of an amino acid residue selected from: an aspartic acid residue, a glutamic acid residue, a histidine residue, an asparagine residue, a glutamine residue, a serine residue, a threonine residue, an alanine residue, a glycine residue, a valine residue, a leucine residue, and an isoleucine residue. The present inventors have identified that amino acid residues from this group represent particularly suitable targets for modification according to the present invention.

In one embodiment, wherein the amino acid modification comprises modification of an amino acid residue selected from an aspartic acid residue, a glutamic acid residue, a histidine residue, an asparagine residue, a glutamine residue, a serine residue, a threonine residue, an alanine residue, a glycine residue, a valine residue, a leucine residue, and an isoleucine residue (as described above), the amino acid residue is substituted with a lysine residue or an arginine residue. In one embodiment, the amino acid residue is substituted with an arginine residue. Thus, in one embodiment, a negatively charged residue, a polar residue, or an uncharged residue is substituted with a positively charged residue, thus increasing the ratio of positive to negative charges and increasing the pi of the clostridial toxin.

In one embodiment, the at least one amino acid modification (as described above) comprises modification of an asparagine amino acid residue or a glutamine amino acid residue (both uncharged, polar residues). In one embodiment, the asparagine or glutamine amino acid residue is substituted with a lysine residue or an arginine residue (both positively charged residues). In one embodiment, the asparagine or glutamine amino acid residue is substituted with a lysine residue. In one embodiment, the asparagine or glutamine amino acid residue is substituted with an arginine residue.

In one embodiment, the engineered clostridial toxin is a BoNT/A. A reference BoNT/A sequence has the UniProtKB Accession Number PI10845. In one embodiment wherein the engineered clostridial toxin is a BoNT/A, the engineered clostridial toxin is a BoNT/A1.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a BoNT/A clostridial toxin H_(N) domain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or all 55) amino acid selected from: D474, N476, D484, N486, 1487, E488, A489, A490, E491, D546, E558, E560, H561, 1566, L568, N570, S571, L577, N578, A597, E599, A601, E620, V621, T623, D625, T631, N645, L647, D650, D651, 1668, E670, A672, V675, S683, 1685, A686, N687, N752, Q753, T755, E756, E757, E758, N760, N761, 1762, N763, D825, 1831, G832, T847, D848, and D858; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15 or all 17) amino acid selected from: N476, S564, N578, E599, L647, D650, D651, V675, 1685, N687, T755, E757, N761, N763, 1831, T847, and 1849; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5 or all 6) amino acid selected from: 5564, L647, D650, D651, T847, and 1849; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5 or all 6) amino acid selected from: N476, N763, N687, E599, 1831, and N761; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, or all 5) amino acid selected from: N578, V675, 1685, T755, and E757; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

In one embodiment, the engineered clostridial toxin is a BoNT/B. A reference BoNT/B sequence has the UniProtKB Accession Number PI0844.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a BoNT/B clostridial toxin H_(N) domain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/B, said engineered BoNT/B comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or all 40) amino acid selected from: V443, G444, D453, S468, D533, E534, N535, T545, L548, D549, 1550, D552, S557, L564, S566, N582, V584, N609, L619, N632, E633, G637, A646, 1655, E657, V662, E669, S670, 1672, D673, N739, 1740, N748, N750, 1818, G819, T834, 1842, N845, and S858; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/B to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/B lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

In one embodiment, the engineered clostridial toxin is a BoNT/Ci. A reference BoNT/Ci sequence has the UniProtKB Accession Number PI8640.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a BoNT/Ci clostridial toxin H_(N) domain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/Ci, said engineered BoNT/Ci comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or all 50) amino acid selected from: L451, D452, C453, E455, V472, T474, D475, L478, N483, E484, E485, E487, 1489, L555, S556, D557, N558, E560, D561, E569, N574, S575, T584, G592, Q594, G596, D617, N640, S641, V642, G645, N646, E661, E665, T667, A670, S678, V680, Q681, E682, S750, G751, S759, Q760, V826, G827, N842, T843, N847, and N853; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/Ci to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/Ci lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

In one embodiment, the engineered clostridial toxin is a BoNT/D. A reference BoNT/D sequence has the UniProtKB Accession Number PI9321.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a BoNT/D clostridial toxin H_(N) domain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/D, said engineered BoNT/D comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or all 62) amino acid selected from: Q469, E470, E473, N474, D479, E480, N482, V483, Q484, N485, S487, D488, S552, N553, N554, V555, E556, N557, 1558, L560, T562, S563, V564, G569, S571, N572, G588, Q590, T614, D616, S619, S622, N636, S637, L639, G641, N642, E657, E661, T663, A666, V669, S674, 1676, Q677, E678, S746, G747, D749, E751, N752, 1753, Q756, N818, V822, G823, E837, N838, T839, N843, N849, and N850; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/D to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/B lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

In one embodiment, the engineered clostridial toxin is a BoNT/E. A reference BoNT/E sequence has the UniProtKB Accession Number Q00496. In one embodiment wherein the engineered clostridial toxin is a BoNT/E, the engineered clostridial toxin is a BoNT/E3.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a BoNT/E clostridial toxin H_(N) domain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E, said engineered BoNT/E comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or all 52) amino acid selected from: D474, N476, E479, E480, D484, N486, I487, E488, A489, A490, E491, E492, L496, D497, Q500, Q501, L504, N507, D509, N510, N514, S516, E518, Q527, L530, N533, 1534, E535, N539, Y548, 1566, L568, D589, A597, E599, A601, L604, Y612, E620, N645, L647, Y648, D651, E737, E741, Y803, Y824, D825, G828, 1831, G832, and D835; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/E to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/E lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

In one embodiment, the engineered clostridial toxin is a BoNT/F. A reference BoNT/F sequence has the UniProtKB Accession Number YP_001390123.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a BoNT/F clostridial toxin H_(N) domain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/F, said engineered BoNT/F comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or all 86) amino acid selected from: N463, E464, N468, T469, D474, D475, T476, T477, N478, N482, N485, N495, 1499, Q501, 1502, Q505, T506, N508, T509, V511, D513, D521, S522, S526, E527, 1528, E529, V534, D535, L536, E549, G550, T552, N553, S558, E566, E567, S568, V586, H587, Q608, D613, A616, D617, S619, N630, N633, N639, E654, V656, E658, L660, T663, L665, V666, S671, 1673, G674, S675, S676, E677, N678, T746, N751, L753, E754, E756, N758, 1759, N760, N761, S799, S821, 1822, N840, S841, E845, L846, S847, S848, T850, N851, D852, 1854, L855, and 1856; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/F to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/F lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

In one embodiment, the engineered clostridial toxin is a BoNT/G. A reference BoNT/G sequence has the UniProtKB Accession Number Q60393.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a BoNT/G clostridial toxin H_(N) domain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/G, said engineered BoNT/G comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or all 36) amino acid selected from: N480, Q482, N483, N484, T485, E487, D540, N562, N570, N571, N572, T588, V589, T615, D621, N637, E638, E642, N643, 1660, E662, 1667, E674, S675, V677, G678, N679, S747, N755, D757, L823, D839, 1841, D844, S846, and L847; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/G to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/G lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

In one embodiment, the engineered clostridial toxin is a TeNT. A reference TeNT sequence has the UniProtKB Accession Number P04958.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a TeNT clostridial toxin H_(N) domain.

In one embodiment, wherein the engineered clostridial toxin is a TeNT, said engineered TeNT comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or all 49) amino acid selected from: A457, S458, L459, D461, L462, E486, E487, Q490, D491, N497, N504, D557, T571, T572, L573, Q574, N580, S581, N588, S589, T590, S598, Q605, G606, Q608, T631, 1633, S640, Q655, E658, G659, N660, E675, 1677, E679, T681, V684, A691, E692, S694, T695, Q696, A772, D773, E774, S862, N866, L867 and D868; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered TeNT to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical TeNT lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a BoNT/A clostridial toxin light chain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/A, said engineered BoNT/A comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 25 or all 28) amino acid selected from: N5, Q7, N9, D12, N15, Q31, D58, N60, D74, N82, T122, D124, E126, Q139, D141, E281, L284, S295, Q31 1, D326, D334, N377, TYR387, N394, N396, N410, M41 1, and N418; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/A to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/A lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a BoNT/B clostridial toxin light chain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/B, said engineered BoNT/B comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40 or all 41 amino acid selected from: N6, N7, N9, Ni 1, D12, N16, N17, N18, D41, E48, E57, N60, D75, D77, N80, E127, N130, N144, E147, E149, E185, N216, D245, E253, N316, D333, E335, D341, N385, D388, N389, E390, E395, E396, D402, D404, E406, E408, Q419, E423, and E427; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/B to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/B lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a BoNT/Ci clostridial toxin light chain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/Ci, said engineered BoNT/Ci comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or all 41) amino acid selected from: N6, N7, N9, D12, D15, N18, N31, E32, N55, N59, N75, N120, N121, N125, D128, Q142, N145, N177, N178, Q183, E184, D208, E21 1, Q247, N255, N31 1, E335, E339, N343, N368, N386, D389, D390, N391, Q396, N405, N407, N425, E427, D442, and N448; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/Ci to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/Ci lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a BoNT/D clostridial toxin light chain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/D, said engineered BoNT/D comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, or all 34) amino acid selected from: D7, N9, D12, N15, D16, N17, D53, D73, D119, E124, E139, E142, N143, Q177, Q178, N180, E184, E255, N308, D335, N336, N339, N343, N368, N386, D389, D390, N391, D397, N403, N407, E409, E416, and N443; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/D to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/D lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a BoNT/E clostridial toxin light chain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E, said engineered BoNT/E comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 35, or all 37) amino acid selected from: N5, N8, N10, D11, N14, D15, Q27, E28, Q53, N72, Q75, D117, N118, D121, N122, Q123, N138, N169, N170, N195, Q237, ILE244, Q290, N293, N297, D312, Q344, N362, N365, D366, N370, E373, N378, N379, N383, N390, and T397; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/E to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/E lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E, said engineered BoNT/E comprises a modification of a least one (for example, at least 1, 2, 3, 4, 5, 10, 15 or 20) amino acid selected from: N5, N8, N10, D11, N14, D15, Q27, E28, N72, Q75, N118, D121, N122, Q123, N138, Q237, Q290, N297, N362, N365, D366, N378, and N379; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/E to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/E lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E, said engineered BoNT/E comprises a modification of a least one (for example, at least 1, 2, 3, 4, 5, 10, 15 or all 19) amino acid selected from: N5, N8, N10, D11, N14, D15, N72, Q75, N118, N122, Q123, N138, Q237, Q290, Q297, N362, D366, N378, and N379; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/E to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/E lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E, said engineered BoNT/E comprises a modification of a least one (for example, at least 1, 2, 3, 4, 5, or all 6) amino acid selected from: N8, N10, Q75, Q123, N138, and Q237; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/E to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/E lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/E, said engineered BoNT/E comprises a modification of a least one (for example, at least 1, 2, or all 3) amino acid selected from: Q123, N138, and Q237; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/E to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/E lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a BoNT/F clostridial toxin light chain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/F, said engineered BoNT/F comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, or all 35) amino acid selected from: N6, N9, Ni 1, D12, N15, D16, D17, E28, D55, D60, D74, N76, E105, E121, N126, E127, N144, D185, N21 1, Q252, N305, E310, D312, N314, N329, D331, N379, D382, D383, D384, E390, N396, N400, D414, and D418; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/F to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/F lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a BoNT/G clostridial toxin light chain.

In one embodiment, wherein the engineered clostridial toxin is a BoNT/G, said engineered BoNT/G comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 35, or all 38) amino acid selected from: N4, N7, N9, Nil, D12, N15, D17, E48, Q55, D57, N60, D75, D127, Q144, E148, D149, Q150, N178, E185, E208, D211, E255, D315, D332, N334, D340, E383, D387, N388, Q393, N394, E395, N403, E407, E418, E422, E426, and N443; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered BoNT/G to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical BoNT/G lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

The present inventors have identified certain amino acids that represent preferred targets for amino acid modification in a TeNT light chain.

In one embodiment, wherein the engineered clostridial toxin is a TeNT, said engineered TeNT comprises a modification of at least one (for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 35, or all 36) amino acid selected from: N6, N7, N15, N16, D17, D31, E51, E57, N60, N76, N101, D126, D143, N167, D179, N180, E251, Q257, N313, N316, D318, D335, N337, Q339, N368, N387, D390, D391, N395, D396, E403, D406, E410, N421, D427, and E450; and said amino acid modification(s) increase(s) the isoelectric point (pi) of the engineered TeNT to a value that is at least 0.2 (for example, at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1) pi units higher than the pi of an otherwise identical TeNT lacking said amino acid modification(s). In one embodiment said modification comprises substitution of the amino acid with a lysine residue or an arginine residue. In one embodiment, said modification comprises substitution of the amino acid with a lysine residue. In one embodiment, said modification comprises substitution of the amino acid with an arginine residue.

The present invention is suitable for application to many different varieties of clostridial toxin. Thus, in the context of the present invention, the term “clostridial toxin” embraces toxins produced by C. botulinum (botulinum neurotoxin serotypes A, B, Ci, D, E, F and G), C. tetani (tetanus neurotoxin), C. butyricum (botulinum neurotoxin serotype E), and C. baratii (botulinum neurotoxin serotype F), as well as modified clostridial toxins or derivatives derived from any of the foregoing. The term “clostridial toxin” also embraces botulinum neurotoxin serotype H.

Botulinum neurotoxin (BoNT) is produced by C. botulinum in the form of a large protein complex, consisting of BoNT itself complexed to a number of accessory proteins. There are at present eight different classes of botulinum neurotoxin, namely: botulinum neurotoxin serotypes A, B, Ci, D, E, F, G, and H, all of which share similar structures and modes of action. Different BoNT serotypes can be distinguished based on inactivation by specific neutralising anti-sera, with such classification by serotype correlating with percentage sequence identity at the amino acid level. BoNT proteins of a given serotype are further divided into different subtypes on the basis of amino acid percentage sequence identity.

BoNTs are absorbed in the gastrointestinal tract, and, after entering the general circulation, bind to the presynaptic membrane of cholinergic nerve terminals and prevent the release of their neurotransmitter acetylcholine. BoNT/B, BoNT/D, BoNT/F and BoNT/G cleave synaptobrevin/vesicle-associated membrane protein (VAMP); BoNT/Ci, BoNT/A and BoNT/E cleave the synaptosomal-associated protein of 25 kDa (SNAP-25); and BoNT/Ci cleaves syntaxin.

Tetanus toxin is produced in a single serotype by C. tetani. C. butyricum produces BoNT/E, while C. baratii produces BoNT/F.

The term “clostridial toxin” is also intended to embrace modified clostridial toxins and derivatives thereof, including but not limited to those described below. A modified clostridial toxin or derivative may contain one or more amino acids that has been modified as compared to the native (unmodified) form of the clostridial toxin, or may contain one or more inserted amino acids that are not present in the native (unmodified) form of the clostridial toxin. By way of example, a modified clostridial toxin may have modified amino acid sequences in one or more domains relative to the native (unmodified) clostridial toxin sequence. Such modifications may modify functional aspects of the toxin, for example biological activity or persistence. Thus, in one embodiment, the engineered clostridial toxin of the invention is an engineered modified clostridial toxin, or an engineered modified clostridial toxin derivative, or an engineered clostridial toxin derivative.

A modified clostridial toxin may have one or more modifications in the amino acid sequence of the heavy chain (such as a modified ¾ domain), wherein said modified heavy chain binds to target nerve cells with a higher or lower affinity than the native (unmodified) clostridial toxin. Such modifications in the He domain can include modifying residues in the ganglioside binding site of the He domain or in the protein (SV2 or synaptotagmin) binding site that alter binding to the ganglioside receptor and/or the protein receptor of the target nerve cell. Examples of such modified clostridial toxins are described in WO 2006/027207 and WO 2006/1 14308, both of which are hereby incorporated by reference in their entirety.

A modified clostridial toxin may have one or more modifications in the amino acid sequence of the light chain, for example modifications in the substrate binding or catalytic domain which may alter or modify the SNARE protein specificity of the modified LC. Examples of such modified clostridial toxins are described in WO 2010/120766 and US 2011/0318385, both of which are hereby incorporated by reference in their entirety.

A modified clostridial toxin may comprise one or more modifications that increases or decreases the biological activity and/or the biological persistence of the modified clostridial toxin. For example, a modified clostridial toxin may comprise a leucine- or tyrosine-based motif, wherein said motif increases or decreases the biological activity and/or the biological persistence of the modified clostridial toxin. Suitable leucine-based motifs include xDxxxLL, xExxxLL, xExxxIL, and xExxxLM (wherein x is any amino acid). Suitable tyrosine-based motifs include Y-x-x-Hy (wherein Hy is a hydrophobic amino acid). Examples of modified clostridial toxins comprising leucine- and tyrosine-based motifs are described in WO 2002/08268, which is hereby incorporated by reference in its entirety.

The term “clostridial toxin” is intended to embrace hybrid and chimeric clostridial toxins. A hybrid clostridial toxin comprises at least a portion of a light chain from one clostridial toxin or subtype thereof, and at least a portion of a heavy chain from another clostridial toxin or clostridial toxin subtype. In one embodiment the hybrid clostridial toxin may contain the entire light chain from one clostridial toxin subtype and the heavy chain from another clostridial toxin subtype. In another embodiment, a chimeric clostridial toxin may contain a portion (e.g. the binding domain) of the heavy chain of one clostridial toxin subtype, with another portion of the heavy chain being from another clostridial toxin subtype. Similarly or alternatively, the therapeutic element may comprise light chain portions from different clostridial toxins. Such hybrid or chimeric clostridial toxins are useful, for example, as a means of delivering the therapeutic benefits of such clostridial toxins to patients who are immunologically resistant to a given clostridial toxin subtype, to patients who may have a lower than average concentration of receptors to a given clostridial toxin heavy chain binding domain, or to patients who may have a protease-resistant variant of the membrane or vesicle toxin substrate (e.g., SNAP-25, VAMP and syntaxin). Hybrid and chimeric clostridial toxins are described in U.S. Pat. No. 8,071,110, which publication is hereby incorporated by reference in its entirety. Thus, in one embodiment, the engineered clostridial toxin of the invention is an engineered hybrid clostridial toxin, or an engineered chimeric clostridial toxin.

The term “clostridial toxin” is intended to embrace re-targeted clostridial toxins. In a re-targeted clostridial toxin, the clostridial toxin is modified to include an exogenous ligand known as a Targeting Moiety (TM). The TM is selected to provide binding specificity for a desired target cell, and as part of the re-targeting process the native binding portion of the clostridial toxin (e.g. the He domain, or the ¾ c domain) may be removed. Re-targeting technology is described, for example, in: EP-B-0689459; WO 1994/021300; EP-B-0939818; U.S. Pat. Nos. 6,461,617; 7,192,596; WO 1998/007864; EP-B-0826051; U.S. Pat. Nos. 5,989,545; 6,395,513; 6,962,703; WO 1996/033273; EP-B-0996468; U.S. Pat. No. 7,052,702; WO 1999/017806; EP-B-1 107794; U.S. Pat. No. 6,632,440; WO 2000/010598; WO 2001/21213; WO 2006/059093; WO 2000/62814; WO 2000/04926; WO 1993/15766; WO 2000/61 192; and WO 1999/58571; all of which are hereby incorporated by reference in their entirety. Thus, in one embodiment, the engineered clostridial toxin of the invention is an engineered re-targeted clostridial toxin.

The present invention also embraces clostridial toxins that have a non-native protease cleavage site. In such clostridial toxins, the native protease cleavage site (also known as the activation site, as described above) is modified or replaced with a protease cleavage site that is not native to that clostridial toxin (i.e. an exogenous cleavage site). Such a site will require an exogenous protease for cleavage, which allows for improved control over the timing and location of cleavage events. Non-native protease cleavage sites that may be employed in clostridial toxins include:

Enterokinase (DDDDKj) Factor Xa (IEGRj/IDGRj) TEV(Tobacco Etch virus) (ENLYFQjG) Thrombin (LVPRjGS) PreScission (LEVLFQjGP).

Additional protease cleavage sites include recognition sequences that are cleaved by a non-cytotoxic protease, for example by the light chain of a clostridial neurotoxin. These include the SNARE (e.g. SNAP-25, syntaxin, VAMP) protein recognition sequences that are cleaved by non-cytotoxic proteases such as the light chain of a clostridial neurotoxin. Clostridial toxins comprising non-native protease cleavage sites are described in U.S. Pat. No. 7,132,259, EP 1206554-B2 and US 2007/0166332, all of which are hereby incorporated by reference in their entirety. Also embraced by the term protease cleavage site is an intein, which is a self-cleaving sequence. The self-splicing reaction is controllable, for example by varying the concentration of reducing agent present.

The present invention also embraces clostridial toxins comprising a “destructive cleavage site”. In said clostridial toxins, a non-native protease cleavage site is incorporated into the clostridial toxin, at a location chosen such that cleavage at said site will decrease the activity of, or inactivate, the clostridial toxin. The destructive protease cleavage site can be susceptible to cleavage by a local protease, in the event that the clostridial toxin, following administration, migrates to a non-target location. Suitable non-native protease cleavage sites include those described above. Clostridial toxins comprising a destructive cleavage site are described in WO 2010/094905 and WO 2002/044199, both of which are hereby incorporated by reference in their entirety.

The engineered clostridial toxins of the present invention, especially the light chain component thereof, may be PEGylated—this may help to increase stability, for example duration of action of the light chain component. PEGylation is particularly preferred when the light chain comprises a BoNT/A, B or Ci protease. PEGylation preferably includes the addition of PEG to the N-terminus of the light chain component. By way of example, the N-terminus of a light chain may be extended with one or more amino acid (e.g. cysteine) residues, which may be the same or different.

One or more of said amino acid residues may have its own PEG molecule attached (e.g. covalently attached) thereto. An example of this technology is described in WO2007/104567, which is hereby incorporated by reference in its entirety.

The engineered clostridial toxins of the present invention may be free from the complexing proteins that are present in a naturally occurring clostridial toxin complex.

An engineered clostridial toxin of the present invention may also comprise a limited number of non-standard amino acids. Thus, in addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline and a-methyl serine) may be substituted for amino acid residues of the engineered clostridial toxins of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for clostridial polypeptide amino acid residues. The engineered clostridial toxins of the present invention can also comprise non-naturally occurring amino acid residues.

Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722. 1991; Ellman et al, Methods Enzymol. 202:301, 1991; Chung et al, Science 259:806-9, 1993; and Chung et al, Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al, J. Biol. Chem. 271:19991-8, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994.

The engineered clostridial toxins of the present invention can be produced using recombinant nucleic acid technologies. Thus, in one embodiment, an engineered clostridial toxin (as described above) is a recombinant engineered clostridial toxin.

In another aspect, the present invention provides a nucleic acid (for example, a DNA) comprising a nucleic acid sequence encoding an engineered clostridial toxin as described above. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector comprising a promoter and a terminator.

In a preferred embodiment, the vector has a promoter selected from:

Promoter Induction Agent Typical Induction Condition Tac (hybrid) IPTG 0.2 mM (0.05-2.0 mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lac operator IPTG 0.2 mM (0.05-2.0 mM)

In another preferred embodiment, the vector has a promoter selected from:

Promoter Induction Agent Typical Induction Condition Tac (hybrid) IPTG 0.2 mM (0.05-2.0 mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lac operator IPTG 0.2 mM (0.05-2.0 mM) T5-lac operator IPTG 0.2 mM (0.05-2.0 mM)

The nucleic acid molecules of the invention may be made using any suitable process known in the art. Thus, the nucleic acid molecules may be made using chemical synthesis techniques. Alternatively, the nucleic acid molecules of the invention may be made using molecular biology techniques.

The DNA construct of the present invention is preferably designed in silico, and then synthesised by conventional DNA synthesis techniques.

The above-mentioned nucleic acid sequence information is optionally modified for codon-biasing according to the ultimate host cell (e.g. E. coli) expression system that is to be employed.

In one embodiment, the nucleic acid sequence encoding an engineered clostridial toxin as described above is a nucleic acid sequence having at least 70% (for example, at least 75, 80, 85, 90, 95, 97, 98 or 99%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 2, 4, and 6. In one embodiment, the nucleic acid sequence has at least 90% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 2, 4, and 6.

The present invention also provides polypeptides encoded by nucleic acid sequences as described above. Thus, in one aspect, the present invention provides a polypeptide comprising an amino acid sequence having at least 70% (for example, at least 75, 80, 85, 90, 95, 97, 98 or 99%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 1, 3 and 5. In one embodiment, the amino acid sequence has at least 90%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 1, 3 and 5.

In one embodiment, the engineered clostridial toxin of the invention is an engineered BoNT/A as described above, and said engineered BoNT/A comprises (or consists of) an amino acid sequence having at least 70% (for example, at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5 or 99.9%) sequence identity to an amino acid sequence selected from SEQ ID NOs: 1, 3 and 5.

In one embodiment, the engineered clostridial toxin of the invention is an engineered BoNT/A as described above, and said engineered BoNT/A comprises (or consists of) the amino acid sequence of SEQ ID NO: 1, 3 or 5.

In one aspect, the invention provides a polypeptide comprising (or consisting of) the amino acid sequence of SEQ ID NO: 1, 3 or 5.

In one aspect, the invention provides a nucleic acid encoding an engineered clostridial toxin as described above, wherein said nucleic acid comprises a nucleic acid sequence having at least 70% (for example, at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5 or 99.9%) sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 2, 4 and 6. In one embodiment, the nucleic acid comprises (or consists of) the nucleic acid sequence of SEQ ID NO: 2, 4 or 6.

In one aspect, the invention provides a nucleic acid comprising (or consisting of) the nucleic acid sequence of SEQ ID NO: 2, 4 or 6.

In one embodiment, the engineered clostridial toxin of the invention is an engineered BoNT/E as described above, and said engineered BoNT/E comprises an amino acid sequence having at least 70% (for example, at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5 or 99.9%) sequence identity to SEQ ID NO: 7.

In one aspect, the invention provides a nucleic acid encoding an engineered clostridial toxin as described above, wherein said nucleic acid comprises a nucleic acid sequence having at least 70% (for example, at least 75, 80, 85, 90, 95, 97, 98, 99, 99.5 or 99.9%) sequence identity to SEQ ID NO: 8.

The “percent sequence identity” between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, % identity may be calculated as the number of identical nucleotides/amino acids divided by the total number of nucleotides/amino acids, multiplied by 100. Calculations of % sequence identity may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimize alignment of two or more sequences. Sequence comparisons and the determination of percent identity between two or more sequences can be carried out using specific mathematical algorithms, such as BLAST, which will be familiar to a skilled person.

In one aspect, the present invention provides a method of producing a single-chain engineered clostridial toxin protein having a light chain and a heavy chain, the method comprising expressing a nucleic acid (said nucleic acid being as described above) in a suitable host cell, lysing the host cell to provide a host cell homogenate containing the single-chain engineered clostridial toxin protein, and isolating the single-chain engineered clostridial toxin protein.

In another aspect, the present invention provides a method of activating an engineered clostridial toxin, the method comprising providing a single-chain engineered clostridial toxin protein obtainable by the method of producing a single-chain engineered clostridial toxin protein as described above, contacting the polypeptide with a protease that cleaves the polypeptide at a recognition site (cleavage site) located between the light chain and heavy chain, thereby converting the polypeptide into a di-chain polypeptide wherein the light chain and heavy chain are joined together by a di sulphide bond.

The engineered clostridial toxins of the invention may be used to prevent or treat certain medical or cosmetic diseases and conditions. Thus, in a further aspect, the present invention provides an engineered clostridial toxin as described above, for use in medicine.

In a related aspect, the present invention provides an engineered clostridial toxin as described above, for use in the prevention or treatment of a disease or condition selected from: strabismus, blepharospasm, squint, dystonia (e.g. spasmodic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g. spasmodic torticollis), beauty therapy (cosmetic) applications benefiting from cell/muscle incapacitation (via SNARE down-regulation or inactivation), neuromuscular disorder or condition of ocular motility (e.g. concomitant strabismus, vertical strabismus, lateral rectus palsy, nystagmus, dysthyroid myopathy), writer's cramp, blepharospasm, bruxism, Wilson's disease, tremor, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, animus, back spasm, charley horse, tension headaches, levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, muscle pain (e.g. pain from muscle spasms), headache pain (e.g. tension headache), brow furrows, skin wrinkles, cancer, uterine disorders, uro-genital disorders, urogenital-neurological disorders, chronic neurogenic inflammation, and a smooth muscle disorder.

In use, the present invention employs a pharmaceutical composition, comprising an engineered clostridial toxin, together with at least one component selected from a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and/or salt.

The engineered clostridial toxins of the present invention may be formulated for oral, parenteral, continuous infusion, inhalation or topical application. Compositions suitable for injection may be in the form of solutions, suspensions or emulsions, or dry powders which are dissolved or suspended in a suitable vehicle prior to use.

In the case of an engineered clostridial toxin that is to be delivered locally, the engineered clostridial toxin may be formulated as a cream (e.g. for topical application), or for sub-dermal injection.

Local delivery means may include an aerosol, or other spray (e.g. a nebuliser). In this regard, an aerosol formulation of an engineered clostridial toxin enables delivery to the lungs and/or other nasal and/or bronchial or airway passages.

Engineered clostridial toxins of the invention may be administered to a patient by intrathecal or epidural injection in the spinal column at the level of the spinal segment involved in the innervation of an affected organ.

A preferred route of administration is via laparoscopic and/or localised, particularly intramuscular, injection.

The dosage ranges for administration of the engineered clostridial toxins of the present invention are those to produce the desired therapeutic effect. It will be appreciated that the dosage range required depends on the precise nature of the engineered clostridial toxin or composition, the route of administration, the nature of the formulation, the age of the patient, the nature, extent or severity of the patient's condition, contraindications, if any, and the judgement of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimisation.

Suitable daily dosages (per kg weight of patient) are in the range 0.0001-1 ng/kg, preferably 0.0001-0.5 ng/kg, more preferably 0.002-0.5 ng/kg, and particularly preferably 0.004-0.5 ng/kg. The unit dosage can vary from less than 1 picogram to 30 ng, but typically will be in the region of 0.01 to 1 ng per dose, which may be administered daily or preferably less frequently, such as weekly or six monthly.

A particularly preferred dosing regimen is based on 0.05 ng of engineered clostridial toxin as the I× dose. In this regard, preferred dosages are in the range 1×-100× (i.e. 0.05-5 ng).

Fluid dosage forms are typically prepared utilising the engineered clostridial toxin and a pyrogen-free sterile vehicle. The engineered clostridial toxin, depending on the vehicle and concentration used, can be either dissolved or suspended in the vehicle. In preparing solutions the engineered clostridial toxin can be dissolved in the vehicle, the solution being made isotonic if necessary by addition of sodium chloride and sterilised by filtration through a sterile filter using aseptic techniques before filling into suitable sterile vials or ampoules and sealing. Alternatively, if solution stability is adequate, the solution in its sealed containers may be sterilised by autoclaving. Advantageously additives such as buffering, solubilising, stabilising, preservative or bactericidal, suspending or emulsifying agents and or local anaesthetic agents may be dissolved in the vehicle.

Dry powders, which are dissolved or suspended in a suitable vehicle prior to use, may be prepared by filling pre-sterilised ingredients into a sterile container using aseptic technique in a sterile area. Alternatively the ingredients may be dissolved into suitable containers using aseptic technique in a sterile area. The product is then freeze dried and the containers are sealed aseptically.

Parenteral suspensions, suitable for intramuscular, subcutaneous or intradermal injection, are prepared in substantially the same manner, except that the sterile components are suspended in the sterile vehicle, instead of being dissolved and sterilisation cannot be accomplished by filtration. The components may be isolated in a sterile state or alternatively it may be sterilised after isolation, e.g. by gamma irradiation.

Advantageously, a suspending agent for example polyvinylpyrrolidone is included in the composition(s) to facilitate uniform distribution of the components.

Administration in accordance with the present invention may take advantage of a variety of delivery technologies including microparticle encapsulation, viral delivery systems or high-pressure aerosol impingement.

FIGURE LEGENDS

FIG. 1.

SDS-PAGE purification of CatH_(N—)v1 (FIG. 1A), CatH_(N—)v2 (FIG. 1B), and CatH_(N—)v3 (FIG. 1C) purification.

FIG. 2.

Percentage SNAP-25 cleavage in rat embryonic spinal cord neurons (eSCN) for CatH_(N_v)1. Rat embryonic spinal cord neurons were cultured for three weeks and treated with CatH_(N—)v1 for 24 h, before Western blotting with SNAP-25 specific antibody. Data is mean±SEM from three independent experiments in triplicate.

FIG. 3.

The potency (t₅ ₀ ) of BoNT/A and CatH_(N—)v1 in the mouse phrenic nerve hemi-diaphragm assay (mPNHD). Data points are individual hemi-diaphragm preparations and means±SEM. CatH_(N—)v1 was statistically significantly slower than reference protein BoNT/A (List Biological Laboratories). 1-way ANOVA and Dunnett's multiple comparisons test. ** p0.01, *** p0.001, **** p0.0001 (1-way ANOVA and Dunnett's multiple comparisons test).

FIG. 4.

Percentage SNAP-25 cleavage in rat embryonic spinal cord neurons (eSCN) for CatH_(N—)v2. Rat embryonic spinal cord neurons were cultured for three weeks and treated with CatH_(N—)v2 for 24 h, before Western blotting with SNAP-25 specific antibody. Data is mean±SEM from three independent experiments in triplicate.

FIG. 5.

The potency (t₅ ₀ ) of BoNT/A and CatH_(N—)v2 in the mouse phrenic nerve hemi-diaphragm assay (mPNHD). Data points are individual hemi-diaphragm preparations and means±SEM. CatH_(N—)v2 is statistically equivalent to the reference protein BoNT/A (List Biological Laboratories). 1-way ANOVA and Dunnett's multiple comparisons test. ** p0.01, *** p0.001, **** p0.0001 (1-way ANOVA and Dunnett's multiple comparisons test).

FIG. 6.

Percentage SNAP-25 cleavage in rat embryonic spinal cord neurons (eSCN) for CatH_(N—)v3. Rat embryonic spinal cord neurons were cultured for three weeks and treated with CatH_(N—)v3 for 24 h, before Western blotting with SNAP-25 specific antibody. Data is mean±SEM from three independent experiments in triplicate.

FIG. 7.

The potency (t₅ ₀ ) of BoNT/A and CatH_(N—)v3 in the mouse phrenic nerve hemi-diaphragm assay (mPNHD). Data points are individual hemi-diaphragm preparations and means±SEM. CatH_(N—)v3 was statistically significantly slower than the reference protein BoNT/A (List Biological Laboratories). 1-way ANOVA and Dunnett's multiple comparisons test. ** p0.01, p0.001, **** p0.0001 (1-way ANOVA and Dunnett's multiple comparisons test).

FIG. 8.

Isoelectric focusing analysis. All three CatH_(N) constructs possess an increased observed pi compared to unmodified BoNT/A.

FIG. 9.

SDS-PAGE purification of CatLC construct.

FIG. 10.

Catalytic activity of CatLC compared to BoNT/E LC reference, with pEC₅o values obtained in the BoTest A/E BoNT Detection Kit (BioSentinal Cat #A1004), following manufacturer's instructions. Data shows mean±standard deviation from one independent experiment in triplicate.

SEQUENCES

-   SEQ ID NO: 1. Engineered BoNT/A, “CatH_(N—)v1”, amino acid sequence. -   SEQ ID NO: 2. Engineered BoNT/A, “CatH_(N—)v1”, nucleic acid     sequence. -   SEQ ID NO: 3. Engineered BoNT/A, “CatH_(N—)v2”, amino acid sequence. -   SEQ ID NO: 4. Engineered BoNT/A, “CatH_(N—)v2”, nucleic acid     sequence. -   SEQ ID NO: 5. Engineered BoNT/A, “CatH_(N—)v3”, amino acid sequence. -   SEQ ID NO: 6. Engineered BoNT/A, “CatH_(N—)v3”, nucleic acid     sequence. -   SEQ ID NO: 7. Engineered BoNT/E light chain, “CatLC”, amino acid     sequence. -   SEQ ID NO: 8. Engineered BoNT/E light chain, “CatLC”, nucleic acid     sequence.

Engineered BoNT/A, “CatH_(N)_vl”, amino acid sequence. SEQ ID NO: 1 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEG DLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERI YSTDLGRMLLTS IVRG IPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVI IGPSADI IQFECKSFGHE VLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHA GHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENE FRLY YYNKFKDIASTLNKAKS IVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLY KMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTI YDGFNLRNTNLAA NFNGQNTEINNMNFTKLKNFTGLFE FYKLLCVRGIITSKTKSLDKGYNKALNDLCIK VNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPE NISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKRRIALTN SVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIA D IT 111PY IGPALNI GNMRYKRRFVGAL IFSGAVI LLEF IPE IA IPVLGT FALVS YIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEAL ENQAEATKAI INYQYNQYTEEEKNNINFNIDDLSSKLNES INKAMININKFLNQCSV SYLMNSMIPYGVKRLEDFDASLKDALLKYI YDNRGTLIGQVDRLKDKVNNTLSRDRPF QLSKYVDNQRLLSTFTEYIKNI INTSILNLRYESNHLIDLSRYASKINIGSKVNFDPI DKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNS ISLNNEYTI IN CMENNSGWKVSLNYGEI IWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRL NNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKE IKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPR GSVMTTNIYLNSSLYRGTKFI IKKYASGNKDNIVRNNDRVYINVWKNKEYRLATNA SQAGVEKILSALEIPDVGNLSQVWMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQ FNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL. Engineered BoNT/A, “CatH_(N)_vl”, nucleic acid sequence. SEQ ID NO: 2 ATGCCATTCGT CAAC AAG CAAT TCAAC TACAAAGAC CCAGTCAAC GGCGTCG ACATCGCATACATCAAGATTCCGAACGCCGGTCAAATGCAGCCGGTTAAGGCTTTTAAG ATCCACAAC AAGA TTTGGGTTATCCCG GAGCGTGACACCTTCACGAAC CCGGAAG AAGGCGATCTGAACGGGCCACGGGAAGCGAAGGAAGTCCGTGTGAGGTAGTAGGATTCG AGGTACCTGAGCACGGATAACGAAAAAGATAAC TACCTGAAAGGTGTGACCAAGCTGT TCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGACTAGCATTGTTCGCGGT ATCCCGTTCTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAAC TGCATTAACGTTATTCAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGGTC ATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAGAGCTTTGGTCACGAG GTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAGTACATTCGTTTTTCGCCG GATTTTACCTTCGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTGCTGGGT GCGGGCAAATTCGCTACCGATCCGGCTGTCACGCTGGCCCATGAACTGATCCACGCA GGCCACCGCCTGTACGGCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACG AATGCATACTACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTC GGTGGCCATGACGCTAAATTCATTGACAGCTTGCAAGAGAATGAGTTCCGTCTGTAC TACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCCAAAAGCATCGTT GGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAGTACCTGCTG TCCGAGGATACCTCCGGCAAGTTTAGCGTTGATAAGCTGAAGTTTGACAAACTGTAC AAGATGCTGACCGAGATTTACACCGAGGACAACTTTGTGAAATTCTTCAAAGTGTTG AATCGTAAAACCTATCTGAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCG AAGGTGAACTACACCATCTATGACGGTTTTAACCTGCGTAACACCAACCTGGCGGCG AACTTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAGTTGAAGAAC TTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGC AAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAAG GTAAACAATTGGGATCTGTTCTTTTCGCCATCCGAAGATAATTTTACCAACGACCTG AACAAGGGTGAAGAAATCACCAGCGATACGAATATTGAAGCAGCGGAAGAGAATATC AGCCTGGATCTGATCCAGCAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAG AACATTAGCATTGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAACTGATGCCG AATATCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTC CATTACCTGCGTGCACAGGAGTTTGAACACGGTAAAcgtCGTATCGCGCTGACCAAC AGCGTTAACGAGGCCCTGCTGAACCCGAGCCGTGTCTATACCTTCTTCAGCAGCGAC TATGTTAAGAAAGTGAACAAAGCCACTGAGGCCGCGATGTTCCTGGGCTGGGTGGAA CAGCTGGTATATGACTTCACGGACGAGACGAGCGAAGTGAGCACTACCGACAAAATT GCTGATATTACCATCATTATCCCGTATATTGGTCCGGCACTGAACATTGGCAACATG CgtTACAAAcgtcgTTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTGCTG GAGTTCATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCTAC ATCGCGAATAAGGTTCTGACGGTTCAGACCATCGATAACGCGCTGTCGAAACGTAAT GAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTGGCTGGCGAAAGTCAAT ACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTGGAGAATCAGGCGGAG GCCACCAAAGCAATTATCAACTACCAATACAACCAGTACACGGAAGAAGAGAAGAAT AACATTAACTTCAATATCGATGATTTGAGCAGCAAGCTGAATGAATCTATCAACAAA GCGATGATCAATATCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAAT AGCATGATTCCGTATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGAT GCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTGATTGGCCAAGTTGACCGC TTGAAAGACAAAGTTAACAATACCCTGAGCcgtGACcgtCCATTTCAACTGAGCAAG TATGTTGATAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATC AATACTAGCATTCTGAACCTGCGTTACGAGAGCAATCATCTGATTGATCTGAGCCGT TATGCAAGCAAGATCAACATCGGTAGCAAGGTCAATTTTGACCCGATCGATAAGAAC CAGATCCAGCTGTTTAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAACGCC ATTGTCTACAACTCCATGTACGAGAATTTCTCCACCAGCTTCTGGATTCGCATCCCG AAATACTTCAACAGCATTAGCCTGAACAACGAGTATACTATCATCAACTGTATGGAG AACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAG GACACCCAAGAGATCAAGCAGCGCGTCGTGTTCAAGTACTCTCAAATGATCAACATT TCCGATTACATTAATCGTTGGATCTTCGTGACCATTACGAATAACCGTCTGAATAAC AGCAAGATTTACATCAATGGTCGCTTGATCGATCAGAAACCGATTAGCAACCTGGGT AATATCCACGCAAG CAACAACATTATGTTCAAAT TGGACGGTTGCCGC GATACC CATCGTTATATCTGGAT CAAGTATTTCAAC CTGTTTGATAAAGAAC TGAAT GAG AAGGAGATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCT GGGGCGATTATCTG CAATACGATAAG CCGTACTATATGCT GAACCTGTATGATCC GAACAAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACATGTATTTGAAGGGT CCGCGTGGCAGCGTTATGACGACCAACATTTACCTGAACTCTAGCCTGTACCGTGGTA CGAAATTCATCAT TAAGAAAT A TGCCAGCGGCAACAAAGAT AACATTGTGCGTA ATAACGATCGTGTCTACATCAACGTGGTCGTGAAGAATAAAGAGTACCGTCTGGCGAC CAACGCTTCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTC GGTAATCTGAGCCAAG TCGTGGTTAT GAAGAG CAAGAAC GAC CAGGGTATCAC TAACAAG TGCAAGAT GAAC CTGCAAGAC AACAAT GGTAAC GACATCGGCTTT ATTGGTTTC CACCAGTTCAACAATA TTGCTAAACTGGTAGCGAGCAATTGGTACAA T CGTCAGATTGAGCGCAGCAGCCGTACTTTGGGCTGTAGCTGGGAGTTTATCCCGGT CGATGATGGTTGGGGCGAACGT C CGCT G. Engineered BoNT/A, “CatH_(N)_v2”, amino acid sequence. SEQ ID NO: 3 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEG DLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERI YSTDLGRMLLTS IVRG IPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVI IGPSADIIQFECKSFGHE VLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHA GHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKF1DSLQENE FRLY YYNKFKDIASTLNKAKS IVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLY KMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTI YDGFNLRNTNLAA NFNGQNTEINNMNFTKLKNFTGLFE FYKLLCVRGI ITSKTKSLDKGYNKALNDLCIK VNNWDLFFSPSEDNFTNDLKKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPE NISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTN SVNEALLNPSRVYTFFSSDYVKKVNKATKAAMFLGWVEQLVYDFTDETSEVSTTDKI ADIT 111PY IGPALNI GNML YKDDFVGAL IFSGAVI LLEFIPE IA IPVLGT FALVSYIAKKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALE NQAEATKAI INYQYNQYTEEEKNKIKFNIDDLSSKLNES INKAMININKFLNQCSVS YLMNSMIPYGVKRLEDFDASLKDALLKYI YDNRGTLKGQVDRLKDKVNNTLSTDIPFQ LSKYVDNQRLLSTFTEYIKNI INTSILNLRYESNHLIDLSRYASKINIGSKVNFDPID KNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNS ISLNNEYTI INC MENNSGWKVSLNYGEI IWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLN NSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKE IKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPR GSVMTTNIYLNSSLYRGTKFI IKKYASGNKDNIVRNNDRVYINVWKNKEYRLATNA SQAGVEKILSALEIPDVGNLSQVWMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQ FNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL. Engineered BoNT/A, “CatH_(N)_v2”, nucleic acid sequence. SEQ ID NO: 4 ATGCCATTCGT CAACAAGCAATTCAACTACAAAGAC CCAGTCAAC GGCGTCGAC ATCGCATACATCAAGATTCCGAACGCCGGTCAAATGCAGCCGGTTAAGGCTTTTAAGAT CCACAACAAGA TTTGGGTTATCCCG GAGCGTGACACCTTCACGAAC CCGGAAGAA G GCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCTGTCAGCTACTACGATTC GACGTACCTGAGCACGGATAAC GAAAAAGAT AAC TACCTGAAAG GTGTGACCAAG CTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGACTAGCATTGTTCG CGGTATCCCGTTCTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTA ACTGCATTAACGTTATTCAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGGTC ATCATTGGCCCGAGCGCAGACATTATCCAATTCGAGTGCAAGAGCTTTGGTCACGAG GTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAGTACATTCGTTTTTCGCCG GATTTTACCTTCGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTGCTGGGT GCGGGCAAATTCGCTACCGATCCGGCTGTCACGCTGGCCCATGAACTGATCCACGCA GGCCACCGCCTGTACGGCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACG AATGCATACTACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTC GGTGGCCATGACGCTAAATTCATTGACAGCTTGCAAGAGAATGAGTTCCGTCTGTAC TACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCCAAAAGCATCGTT GGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAGTACCTGCTG TCCGAGGATACCTCCGGCAAGTTTAGCGTTGATAAGCTGAAGTTTGACAAACTGTAC AAGATGCTGACCGAGATTTACACCGAGGACAACTTTGTGAAATTCTTCAAAGTGTTG AATCGTAAAACCTATCTGAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCG AAGGTGAACTACACCATCTATGACGGTTTTAACCTGCGTAACACCAACCTGGCGGCG AACTTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAGTTGAAGAAC TTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGC AAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAAG GTAAACAATTGGGATCTGTTCTTTTCGCCATCCGAAGATAATTTTACCAACGACCTG AAgAAGGGTGAAGAAATCACCAGCGATACGAATATTGAAGCAGCGGAAGAGAATATC AGCCTGGATCTGATCCAGCAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAG AACATTAGCATTGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAACTGATGCCG AATATCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTC CATTACCTGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGCGCTGACCAAC AGCGTTAACGAGGCCCTGCTGAACCCGAGCCGTGTCTATACCTTCTTCAGCAGCGAC TATGTTAAGAAAGTGAACAAAGCCACTaAGGCCGCGATGTTCCTGGGCTGGGTGGAA CAGCTGGTATATGACTTCACGGACGAGACGAGCGAAGTGAGCACTACCGACAAAATT GCTGATATTACCATCATTATCCCGTATATTGGTCCGGCACTGAACATTGGCAACATG CTGTACAAAGACGATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTGCTG GAGTTCATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCTAC ATCGCGAAgAAGGTTCTGACGGTTCAGACCATCGATAACGCGCTGTCGAAACGTAAT GAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTGGCTGGCGAAAGTCAAT ACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTGGAGAATCAGGCGGAG GCCACCAAAGCAATTATCAACTACCAATACAACCAGTACACGGAAGAAGAGAAGAAT AAgATTAAgTTCAATATCGATGATTTGAGCAGCAAGCTGAATGAATCTATCAACAAA GCGATGATCAATATCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAAT AGCATGATTCCGTATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGAT GCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTGAagGGCCAAGTTGACCGC TTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCATTTCAACTGAGCAAG TATGTTGATAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATC AATACTAGCATTCTGAACCTGCGTTACGAGAGCAATCATCTGATTGATCTGAGCCGT TATGCAAGCAAGATCAACATCGGTAGCAAGGTCAATTTTGACCCGATCGATAAGAAC CAGATCCAGCTGTTTAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAACGCC ATTGTCTACAACTCCATGTACGAGAATTTCTCCACCAGCTTCTGGATTCGCATCCCG AAATACTTCAACAGCATTAGCCTGAACAACGAGTATACTATCATCAACTGTATGGAG AACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAG GACACCCAAGAGATCAAGCAGCGCGTCGTGTTCAAGTACTCTCAAATGATCAACATT TCCGATTACATTAATCGTTGGATCTTCGTGACCATTACGAATAACCGTCTGAATAAC AGCAAGATTTACATCAATGGTCGCTTGATCGATCAGAAACCGATTAGCAACCTGGGT AATATCCACGCAAGCAACAACATTATGTTCAAATTGGACGGTTGCCGCGATACCCAT CGTTATATCTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTGAATGAGAAGGAG ATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCTGGGGC GATTATCTGCAATACGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAA TATGTGGATGTCAATAATGTGGGTATTCGTGGTTACATGTATTTGAAGGGTCCGCGT GGCAGCGTTATGACGACCAACATTTACCTGAACTCTAGCCTGTACCGTGGTACGAAA TTCATCATTAAGAAATATGCCAGCGGCAACAAAGATAACATTGTGCGTAATAACGAT CGTGTCTACATCAACGTGGTCGTGAAGAATAAAGAGTACCGTCTGGCGACCAACGCT TCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAAT CTGAG CCAAG TCGTGGTTAT GAAGAG CAAGAAC GAC CAGGGTA TCACTAA C AAG TGCAAGAT GAAC CTGCAAGAC AAC AAT GGTAAC GACA TCGGCTT TATTGGTTTC CACCAGTTCAAC AAT A TTGCTAAAC TGGTAG CGAG CAAT TGGTA CAAT CGTCAGAT TGAGCGCAGCAGCCGTACTTTGGGCTGTAGCTGGGAG TTTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTG. Engineered BoNT/A, “CatH_(N)_v3”, amino acid sequence. SEQ ID NO: 5 MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEG DLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERI YSTDLGRMLLTS IVRG IPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVI IGPSADI IQFECKSFGHE VLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHA GHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENE FRLY YYNKFKDIASTLNKAKS IVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLY KMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTI YDGFNLRNTNLAA NFNGQNTEINNMNFTKLKNFTGLFE FYKLLCVRGI ITSKTKSLDKGYNKALNDLCIK VNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPE NIS IENLSSDI IGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTN SVNEALLKPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKI ADITI IIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPKLGTFALVSY KANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAE ATKAI INYQYNQYKEKEKNNINFNIDDLSSKLNES INKAMININKFLNQCSVSYLMN SMIPYGVKRLEDFDASLKDALLKYI YDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSK YVDNQRLLSTFTEYIKNI INTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKN QIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNS ISLNNEYTI INCME NNSGWKVSLNYGEIIWTLQDTQEIKQRWFKYSQMINISDYINRWIFVTITNNRLNN SKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKE IKDLYDNQSNSGILKDFWGDYLQYDKP YYMLNLYDPNKYVDVNNVGIRGYMYLKGPR GS\/MTTNI YLNSSLYRGTKFI IKKYASGNKDNIVRNNDRVYIN\AA/KNKEYRLATNA SQAGVEKILSALEIPDVGNLSQ\AA/MKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQ FNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL. Engineered BoNT/A, “CatH_(N)_v3”, nucleic acid sequence. SEQ ID NO: 6 ATGCCATTCGT CAAC AAG CAAT TCAAC TACAAAGAC CCAGTCAAC GGCGTCG ACATCGCATACATCAAGATTCCGAACGCCGGTCAAATGCAGCCGGTTAAGGCTTTTAAG ATCCA CAAC AAG A TTTGGGTTATCCCG GAGCGTGACACCTTCACGAAC CCGGAA G AAG GCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCTGTCAGCTACTACGATT CGACGTACCTGAG CACGGATAAC G AAA AAGAT AAC TACCTGAAAG GTGTGACC AAG CTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCTGCTGACTAGCATTGT TCGCGGTATCCCGTTCTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACAC TAACTGCATTAACGTTATTCAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGGTC A TCAT TGGCCCGAG CGCAGAC A TTA TCCAAT TCGAG TGCAAGAG CTTTGGTC A CGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAGTACATTCGTTTTTCGC CGGATTTTACCTTCGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTGCTGGGT GCGGGCAAATTCGCTACCGATCCGGCTGTCACGCTGGCCCATGAACTGATCCACGCA GGCCACCGCCTGTACGGCATTGCCATCAACCCAAACCGTGTGTTCAAGGTTAATACG AATGCATACTACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTC GGTGGCCATGACGCTAAATTCATTGACAGCTTGCAAGAGAATGAGTTCCGTCTGTAC TACTATAACAAATTCAAAGACATTGCAAGCACGTTGAACAAGGCCAAAAGCATCGTT GGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAGTACCTGCTG TCCGAGGATACCTCCGGCAAGTTTAGCGTTGATAAGCTGAAGTTTGACAAACTGTAC AAGATGCTGACCGAGATTTACACCGAGGACAACTTTGTGAAATTCTTCAAAGTGTTG AATCGTAAAACCTATCTGAATTTTGACAAAGCGGTTTTCAAGATTAACATCGTGCCG AAGGTGAACTACACCATCTATGACGGTTTTAACCTGCGTAACACCAACCTGGCGGCG AACTTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAGTTGAAGAAC TTCACGGGTCTGTTCGAGTTCTATAAGCTGCTGTGCGTGCGCGGTATCATCACCAGC AAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTGAATGACCTGTGCATTAAG GTAAACAATTGGGATCTGTTCTTTTCGCCATCCGAAGATAATTTTACCAACGACCTG AACAAGGGTGAAGAAATCACCAGCgAtACGAATATTGAAGCAGCGGAAGAGAATATC AGCCTGGATCTGATCCAGCAGTACTATCTGACCTTTAACTTCGACAATGAACCGGAG AACATTAGCATTGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAACTGATGCCG AATATCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTC CATTACCTGCGTGCACAGGAGTTTGAACACGGTAAAAGCCGTATCGCGCTGACCAAC AGCGTTAACGAGGCCCTGCTGAAaCCGAGCCGTGTCTATACCTTCTTCAGCAGCGAC TATGTTAAGAAAGTGAACAAAGCCACTGAGGCCGCGATGTTCCTGGGCTGGGTGGAA CAGCTGGTATATGACTTCACGGACGAGACGAGCGAAGTGAGCACTACCGACAAAATT GCTGATATTACCATCATTATCCCGTATATTGGTCCGGCACTGAACATTGGCAACATG CTGTACAAAGACGATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTGCTG GAGTTCATTCCGGAGATTGCGATCCCGaaGTTGGGTACCTTCGCGCTGGTGTCCTAC AagGCGAATAAGGTTCTGACGGTTCAGACCATCGATAACGCGCTGTCGAAACGTAAT GAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTGGCTGGCGAAAGTCAAT ACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTGGAGAATCAGGCGGAG GCCACCAAAGCAATTATCAACTACCAATACAACCAGTACAaGGAAaAAGAGAAGAAT AACATTAACTTCAATATCGATGATTTGAGCAGCAAGCTGAATGAATCTATCAACAAA GCGATGATCAATATCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAAT AGCATGATTCCGTATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGAT GCGTTGCTGAAATACATTTACGACAATCGTGGTACGCTGATTGGCCAAGTTGACCGC TTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCATTTCAACTGAGCAAG TATGTTGATAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATC AATACTAGCATTCTGAACCTGCGTTACGAGAGCAATCATCTGATTGATCTGAGCCGT TATGCAAGCAAGATCAACATCGGTAGCAAGGTCAATTTTGACCCGATCGATAAGAAC CAGATCCAGCTGTTTAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAACGCC ATTGTCTACAACTCCATGTACGAGAATTTCTCCACCAGCTTCTGGATTCGCATCCCG AAATACTTCAACAGCATTAGCCTGAACAACGAGTATACTATCATCAACTGTATGGAG AACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTTGCAG GACACCCAAGAGATCAAGCAGCGCGTCGTGTTCAAGTACTCTCAAATGATCAACATT TCCGATTACATTAATCGTTGGATCTTCGTGACCATTACGAATAACCGTCTGAATAAC AGCAAGATTTACATCAATGGTCGCTTGATCGATCAGAAACCGATTAGCAACCTGGGT AATATCCACGCAAGCAACAACATTATGTTCAAATTGGACGGTTGCCGCGATACCCAT CGTTATATCTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTGAATGAGAAGGAG ATCAAAGATTTGTATGACAACCAATCTAACAGCGGCATTTTGAAGGACTTCTGGGGC GATTATCTGCAATACGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAACAAA TATGTGGATGTCAATAATGTGGGTATTCGTGGTTACATGTATTTGAAGGGTCCGCGT GGCAGCGTTATGACGACCAACATTTACCTGAACTCTAGCCTGTACCGTGGTACGAAA TTCATCATTAAGAAATATGCCAGCGGCAACAAAGATAACATTGTGCGTAATAACGAT CGTGTCTACATCAACGTGGTCGTGAAGAATAAAGAGTACCGTCTGGCGACCAACGCT TCGCAGGCGGGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAAT CTGAGCCAAGTCGTGGTTATGAAGAGCAAGAACGACCAGGGTATCACTAACAAGTGC AAGATGAACCTGCAAGACAACAATGGTAACGACATCGGCTTTATTGGTTTCCACCAG TTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTACAATCGTCAGATTGAGCGC AGCAGCCGTACTTTGGGCTGTAGCTGGGAGTTTATCCCGGTCGATGATGGTTGGGGC GAACGTCCGCTG. Engineered BoNT/E light chain, “CatLC”, amino acid sequence. SEQ ID NO: 7 MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPD IIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLI YNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENG KYD IKDVGVDNAGAKAGL TFLVDL IKNKIIMNADT DY S IAEAAFNKGE TAMT INGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYL LTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWY AVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISEFGSMPKINS FNYNDPVNDRTILYIKPGGCQEFYKSFNIMKNIWI IPERNVIGTTPQDFHPPTSLKN GDSSYYDPNYLQSDEEKDRFLKIVTKI FNRINNNLSGGILLEELSKANPYLGNDNTP DNKFHIGDASAVEIKFSKGSQHTLLPNVT IMGAEPDLFETNSSNISLRNNYMPSNHG FGS IAIVTFSPEYSFRFNDNS INEFIQDPALTLMHELIHSLHGLYGAKGITTTCI ITQQKNPLITNRKGINIEEFLTFGGNDLNIITVAQYNDIYTNLLNDYRKIASKLSKVQV SNPQLNPYKDI FQEKYGLDKDASGI YSVNINKFDDILKKLYSFTEFDLATKFQVKCR ETYIGQYKYFKLSNLLNDS IYNISEGYNINNLKVNFRGQNANLNPRI IKPITGRGLV KKI IRFAVDKLAAALEHHHHHH. Engineered BoNT/E light chain, “CatLC”, amino acid sequence. SEQ ID NO: 8 A TGAAAAT CGAAGAAG GTAAAC TGGTAAT CTGGAT TAAC GGCGATAAAG GC TAT AACGGTCTCGCTGAAGTCGGTAAGAAAT TCGAGAAAGATACCGGAAT TAAAGT CACCGTTGAGCATCCGGATAAACTGGAAGAGAAATTCCCACAGGTTGCGGCAACTGGCG ATGGCCCTGACATTATCTTCTGGGCACACGACCGCTTTGGTGGCTACGCTCAATCTGGC CTGTTGGCTGAAATCACCCCGGACAAAGCGTTCCAGGACAAGCTGTATCCGTTTACCTG GGATGCCGTACGTTACAACGGCAAGCTGATTGCTTACCCGATCGCTGTTGAAGCGTTAT CGCTGAT TTATAACAAAGATCTGCTGCC GAAC CCGCCAAAAACCTGGGAAGAGATC CCGGCGCTGGATAAAGAACTGAAAGCGAAAGGTAAGAGCGCGCTGATGTTCAACCTGCA AGAACCGTACTTCACCTGGCCGCTGATTGCTGCTGACGGGGGTTATGCGTTCAAGTATG AAAACGGCAAGTACGACATTAAAGACGTGGGCGTGGATAACGCTGGCGCGAAAGCGGG TCTGACCTTCCTGGTTGACCT GAT TAAAAACAAACACAT GAAT GCAGACACCGAT TACTCCAT CGCAGAAG CTGCCTTTAATAAAG GCGAAACAGCGAT GACCAT CAA CGGCCCGTGGGCATGGTCCAACATCGACACCAGCAAAGTGAATTATGGTGTAACGGTA CTGCCGACCTTCAAGGGTCAACCATCCAAACCGTTCGTTGGCGTGCTGAGCGCAGGT ATTAACGCCGCCAGTCCGAACAAAGAGCTGGCAAAAGAGTTCCTCGAAAACTATCTG CTGACTGATGAAGGTCTGGAAGCGGTTAATAAAGACAAACCGCTGGGTGCCGTAGCG CTGAAGTCTTACGAGGAAGAGTTGGCGAAAGATCCACGTATTGCCGCCACTATGGAA AACGCCCAGAAAGGTGAAATCATGCCGAACATCCCGCAGATGTCCGCTTTCTGGTAT GCCGTGCGTACTGCGGTGATCAACGCCGCCAGCGGTCGTCAGACTGTCGATGAAGCC CTGAAAGACGCGCAGACTAATTCGAGCTCGAACAACAACAACAATAACAATAACAAC AACCTCGGGATCGAGGGAAGGATTTCAGAATTCGGATCCATGCCAAAAATCAACAGC TTTAATTACAATGACCCTGTAAACGATCGTACCATCCTATACATAAAGCCGGGTGGG TGTCAAGAGTTCTACAAATCTTTCAATATTATGAAGAATATATGGATTATACCTGAG CGTAACGTTATTGGTACGACACCGCAAGATTTTCATCCACCTACTTCGTTGAAGAAC GGTGACTCTTCCTATTACGACCCCAATTATCTCCAGTCGGATGAAGAGAAGGACAGA TTCCTTAAAATAGTAACCAAAATCTTTAACAGGATTAATAACAATCTATCCGGAGGT ATTTTGCTTGAAGAGCTTAGTAAAGCTAATCCTTACCTAGGTAACGATAATACACCA CACAACAAGTTTCATATAGGCGATGCATCCGCCGTGGAAATCAAATTTAGCAAGGGA TCACAGCATATTCTCTTGCCCAACGTTATTATAATGGGGGCGGAACCAGATTTATTT GAAACAAATTCGAGTAATATTAGCCTGAGAAATAACTATATGCCGTCAAACCATGGG TTCGGTAGCATAGCGATCGTTACTTTTTCTCCCGAATACAGTTTTCGCTTCAATGAT AATAGTATAAATGAGTTTATCCAAGACCCCGCACTCACGCTTATGCACGAACTCATA CACTCTTTACACGGCCTGTATGGCGCTAAGGGGATAACCACTACGTGTATCATTACT CAGCAAAAGAACCCATTGATAACGAACAGGAAGGGCATTAACATCGAGGAATTTCTT ACATTTGGAGGCAACGATCTGAACATTATAACTGTCGCACAGTACAATGACATCTAT ACCAACTTACTAAATGATTATAGAAAAATCGCTTCTAAGTTATCCAAGGTTCAAGTC TCAAACCCTCAACTGAATCCGTATAAGGACATATTCCAAGAGAAATATGGATTAGAC AAAGACGCGTCAGGAATCTATTCGGTAAACATTAACAAATTCGACGATATTTTGAAG AAACTTTACAGCTTCACGGAGTTCGACTTGGCCACCAAATTCCAGGTCAAATGCCGA GAGACATACATCGGACAGTATAAGTATTTCAAGCTGTCGAATCTCCTGAATGATTCC ATATACAACATTAGTGAGGGTTACAATATAAATAACCTAAAGGTGAATTTCCGAGGC CAAAACGCCAACCTAAATCCGCGCATCATTAAACCCATCACAGGACGGGGGTTAGTG AAGAAAATAATCCGGTTTGCGGTCGACAACCTTCCGGCCGCACTCCAGCACCACCAC CACCACCAC.

EXAMPLES

The following Examples serve to illustrate particular embodiments of the invention, and do not limit the scope of the invention defined in the claims in any way.

Example 1 Identification of Preferred Clostridial Toxin Amino Acids for Modification.

The amino acids identified as suitable candidates for modification (mutation sites) were selected using a number of different criteria.

1. Location of residue within BoNT molecule (within H_(N), excluding belt region) 2. Location with regard to secondary/tertiary structure; 3. Type of residue; 4. Degree of surface exposure.

Acidic, neutral, polar and hydrophobic residues were considered for selection.

Exposed residues were determined using ArealMol from the CCP4 suite. Each structure was analysed by ArealMol, and exposed residues were identified as having a sum value greater than 55.

Secondary structures within the H_(N) of each Subtype and TeNT were identified using a secondary structure assignment program (Stride Web Interface). Regions assigned as forming α-helix, β-strand or 3i₀-helix were excluded from the selection.

Sequences Used

Accession numbers:

BoNT/A: PI0845 BoNT/B: PI0844 BoNT/C₁: PI8640 BoNT/D: PI9321 BoNT/E: Q00496 BoNT/F: YP_001390123 BoNT/G: Q60393 Structural Data Source

Crystal structures of BoNT/A (3BTA.pdb), BoNT/B (1EPW), and BoNT/E (3FFZ.pdb) obtained from RCSB. Homology modelling of BoNT/Ci, BoNT/D, BoNT/F, and BoNT/G performed using LOOPP and the following sequences, respectively: PI8640, PI9321, YP_001390123, and Q60393.

Preferred clostridial toxin amino acid residues for modification in the clostridial toxin H_(N) domain:

BoNT/A:

D474, N476, D484, N486, 1487, E488, A489, A490, E491, D546, E558, E560, H561, 1566, L568, N570, S571, L577, N578, A597, E599, A601, E620, V621, T623, D625, T631, N645, L647, D650, D651, 1668, E670, A672, V675, S683, 1685, A686, N687, N752, Q753, T755, E756, E757, E758, N760, N761, 1762, N763, D825, 1831, G832, T847, D848, and D858

BoNT/B:

V443, G444, D453, S468, D533, E534, N535, T545, L548, D549, 1550, D552, S557, L564, S566, N582, V584, N609, L619, N632, E633, G637, A646, 1655, E657, V662, E669, S670, 1672, D673, N739, 1740, N748, N750, 1818, G819, T834, 1842, N845, and S858

BoNT/C₁:

L451, D452, C453, E455, V472, T474, D475, L478, N483, E484, E485, E487, 1489, L555, S556, D557, N558, E560, D561, E569, N574, S575, T584, G592, Q594, G596, D617, N640, S641, V642, G645, N646, E661, E665, T667, A670, S678, V680, Q681, E682, S750, G751, S759, Q760, V826, G827, N842, T843, N847, and N853

BoNT/D:

Q469, E470, E473, N474, D479, E480, N482, V483, Q484, N485, S487, D488, S552, N553, N554, V555, E556, N557, 1558, L560, T562, S563, V564, G569, S571, N572, G588, Q590, T614, D616, S619, S622, N636, S637, L639, G641, N642, E657, E661, T663, A666, V669, S674, 1676, Q677, E678, S746, G747, D749, E751, N752, 1753, Q756, N818, V822, G823, E837, N838, T839, N843, N849, and N850

BoNT/E:

D474, N476, E479, E480, D484, N486, 1487, E488, A489, A490, E491, E492, L496, D497, Q500, Q501, L504, N507, D509, N510, N514, S516, E518, Q527, L530, N533, 1534, E535, N539, Y548, 1566, L568, D589, A597, E599, A601, L604, Y612, E620, N645, L647, Y648, D651, E737, E741, Y803, Y824, D825, G828, 1831, G832, and D835

BoNT/F:

N463, E464, N468, T469, D474, D475, T476, T477, N478, N482, N485, N495, 1499, Q501, 1502, Q505, T506, N508, T509, V51 1, D513, D521, S522, S526, E527, 1528, E529, V534, D535, L536, E549, G550, T552, N553, S558, E566, E567, S568, V586, H587, Q608, D613, A616, D617, S619, N630, N633, N639, E654, V656, E658, L660, T663, L665, V666, S671, 1673, G674, S675, S676, E677, N678, T746, N751, L753, E754, E756, N758, 1759, N760, N761, S799, S821, 1822, N840, S841, E845, L846, S847, S848, T850, N851, D852, 1854, L855, and 1856

BoNT/G:

N480, Q482, N483, N484, T485, E487, D540, N562, N570, N571, N572, T588, V589, T615, D621, N637, E638, E642, N643, 1660, E662, 1667, E674, S675, V677, G678, N679, S747, N755, D757, L823, D839, 1841, D844, S846, and L847

TeNT:

A457, S458, L459, D461, L462, E486, E487, Q490, D491, N497, N504, D557, T571, T572, L573, Q574, N580, S581, N588, S589, T590, S598, Q605, G606, Q608, T631, 1633, S640, Q655, E658, G659, N660, E675, 1677, E679, T681, V684, A691, E692, S694, T695, Q696, A772, D773, E774, S862, N866, L867 and D868

Preferred clostridial toxin amino acid residues for modification in the BoNT/E light chain:

N5, N8, N10, D11, N14, D15, Q27, E28, N72, Q75, N118, D121, N122, Q123, N138, Q237, Q290, N297, N362, N365, D366, N378, and N379.

Example 2 Design of Engineered BoNT/a Molecules

Three different examples of an engineered BoNT/A molecule according to the present invention were produced.

Using the method described in Example 1, a total of 55 residues were identified as candidates for mutation in the BoNT/A H_(N) domain. The suitability of the residues was further assessed by visual inspection of the BoNT/A crystal structure to give a list of 11 preferred candidates (N476, N763, N687, E599, 183 1, N761, N578, V675, 1685, T755, E757). A further six residues were chosen based on functional data that showed that these residues were amenable to mutation without adversely affecting protein function. Four of these residues were within the candidate list (L647, D650, D65 1, T847) and two were not (S564, 1849). With the 11 residues from the candidate list plus the six from functional data, a total of 17 residues were selected for mutation.

From the 17 residues selected, 3 constructs were made: CatH_(N_v)1, CatH_(N_v)2 and CatH_(N_v)3. The mutations for the CatH_(N) constructs are shown in Table 3 below:

TABLE 3 CatH_(N) constructs with mutations listed and calculated pi Number of Calculated Calculated ΔpI relative Mutations Mutations pI to BoNT/A (pI 6.4) CatHN_v1 S564R, 6 7.4 1.0 L647R, D650R. D651R, T847R, I849R CatHN_v2 N476K, 6 7.3 0.9 N763K, N687K, E599K, 183IK, N761K CatHN_v3 N578K, 5 7.1 0.7 V675K, I685K, T755K, E757K [ΔpI = change in isoelectric point]

Purification of CatH_(N_v)1, CatH_(N_v)2 and CatH_(N_v)3 is shown in FIGS. 1A, 1B and 1C, respectively.

Example 3 Cloning, Expression and Purification

DNA constructs encoding the engineered BoNT/A molecules described in Example 2 were synthesised, cloned into the pJ401 expression vector and then transformed into BL21 (DE3) E. coli. This allowed for soluble over-expression of the recombinant engineered BoNT/A molecules in E. coli.

The recombinant engineered BoNTs were purified using classical chromatography techniques from the E. coli lysates. An initial purification step using a cation-exchange resin was employed, followed by an intermediate purification step using a hydrophobic interaction resin. The recombinant engineered BoNT single-chain was then cleaved by proteolysis, resulting in the activated di-chain engineered BoNT. A final purification step was then employed to remove remaining contaminants.

Example 4 Characterization of Purified Engineered BoNTs

The engineered BoNTs described in Example 2 above were characterised experimentally.

The ability of the engineered BoNTs to enter neurons and cleave SNAP-25 (the target of BoNT/A) was assessed using rat embryonic spinal cord neurons (eSCN). Potency of the engineered BoNTs was further assessed using the mouse phrenic nerve hemi-diaphragm assay (mPNHD).

CatH_(N_v)1:

The first set of mutations added were substitutions to Arginine:

S564R, L647R, D650R, D65 IR, T847R, I849R

The CatH_(N_v)1 molecule was tested in the rat embryonic spinal cord neuron (eSCN) SNAP-25 cleavage assay, and found to be equipotent potent to BoNT/A (BoNT/A) (FIG. 2).

A positive result was also demonstrated in the mouse phrenic nerve hemi-diaphragm (mPNHD) assay (FIG. 3).

CatH_(N_v)2:

The second set of mutations were substitutions to Lysine:

N476K, N763K, N687K, E599K, 183 1K, N761K

The CatH_(N_v)2 protein was tested in the eSCN SNAP-25 cleavage assay, and found to retain the ability to enter the cells and cleave SNAP-25. In the mPNHD assay CatH_(N_v)2 was equipotent to BoNT/A (FIGS. 4 and 5).

CatH_(N_v)3:

The third set of mutations were substitutions to Lysine:

N578K, V675K, 1685K, T755K, E757K

The CatH_(N_v)3 molecule was tested in the eSCN SNAP-25 cleavage assay, and found to retain the ability to enter the cells and cleave SNAP-25. Similarly, a positive result was also demonstrated in the mPNHD assay (FIGS. 6 and 7).

Isoelectric Focusing

All three CatH_(N) constructs possess an increased pi compared to unmodified BoNT/A (FIG. 8).

Example 5 Modifications in the Light Chain of BoNT/E

Due to the modularity of the botulinum toxin, a BoNT/E light chain construct with an N-terminal maltose binding protein (MBP) tag and a C-terminal 6 histidine tag (6HT) was used as a surrogate to assay BoNT/E activity when mutated and characterised.

A BoNT/E light chain construct (“CatLC”) was prepared having the mutations shown in table below.

TABLE 4 Construct with mutations listed, calculated pi and calculated ΔpI . Number of Calculated Calculated ΔpI relative Mutations Mutations pI to MBP-LC/E (pI 6.3) CatLC Q123K 3 6.6 0.3 N138K Q237K

The construct was expressed in BL21 DE3 cells and purified using affinity chromatography. Purification is shown in FIG. 9.

Assessment of the construct for catalytic activity showed that the modified light chain retained the catalytic activity of unmodified, light chain (FIG. 10). 

1. An engineered clostridial toxin comprising at least one amino acid modification, wherein said at least one amino acid modification increases the isoelectric point (pi) of the engineered clostridial toxin to a value that is at least 0.2 pi units higher than the pi of an otherwise identical clostridial toxin lacking said at least one amino acid modification, and wherein said at least one amino acid modification is not located in the clostridial toxin binding domain (¾ domain).
 2. The engineered clostridial toxin of claim 1, wherein said at least one amino acid modification is located in the clostridial toxin translocation domain (H_(N) domain).
 3. The engineered clostridial toxin of claim 1, wherein said at least one amino acid modification is located in the clostridial toxin light chain.
 4. The engineered clostridial toxin of claim 3, wherein said at least one amino acid modification does not introduce into the clostridial toxin light chain an E3 ligase recognition motif.
 5. The engineered clostridial toxin of any one of claims 1 to 4, wherein said at least one amino acid modification increases the isoelectric point (pi) of the engineered clostridial toxin to a value that is at least 0.5 pi units higher than the pi of an otherwise identical clostridial toxin lacking said at least one amino acid modification.
 6. The engineered clostridial toxin of any one of claims 1 to 4, wherein said at least one amino acid modification increases the isoelectric point (pi) of the engineered clostridial toxin to a value that is at least one pi unit higher than the pi of an otherwise identical clostridial toxin lacking said at least one amino acid modification.
 7. The engineered clostridial toxin of any one of claims 1 to 4, wherein said at least one amino acid modification increases the isoelectric point (pi) of the engineered clostridial toxin to a value that is at least two pi units higher than the pi of an otherwise identical clostridial toxin lacking said at least one amino acid modification.
 8. The engineered clostridial toxin of any one of claims 1 to 4, wherein said at least one amino acid modification increases the isoelectric point (pi) of the engineered clostridial toxin to a value that is between 2 and 5 pi units higher than the pi of an otherwise identical clostridial toxin lacking said at least one amino acid modification.
 9. The engineered clostridial toxin of any preceding claim, wherein the engineered clostridial toxin has a pi of at least 6.5.
 10. The engineered clostridial toxin of any preceding claim, wherein the engineered clostridial toxin has a pi of between 6.5 and 9.5.
 11. The engineered clostridial toxin of any preceding claim, wherein the engineered clostridial toxin has a pi of between 6.5 and 7.5.
 12. The engineered clostridial toxin of any preceding claim, wherein the at least one amino acid modification is selected from: an amino acid substitution, an amino acid insertion, and an amino acid deletion.
 13. The engineered clostridial toxin of claim 12, wherein the at least one amino acid substitution is selected from: substitution of an acidic amino acid residue with a basic amino acid residue, substitution of an acidic amino acid residue with an uncharged amino acid residue, and substitution of an uncharged amino acid residue with a basic amino acid residue.
 14. The engineered clostridial toxin of any previous claim, wherein the engineered clostridial toxin comprises between 1 and 90 amino acid modifications.
 15. The engineered clostridial toxin of any preceding claim, wherein the engineered clostridial toxin comprises at least three amino acid modifications.
 16. The engineered clostridial toxin of any preceding claim, wherein the engineered clostridial toxin comprises between 4 and 40 amino acid modifications.
 17. The engineered clostridial toxin of any preceding claim, wherein the at least one amino acid modification is a modification of a surface exposed amino acid residue.
 18. The engineered clostridial toxin of any preceding claim, wherein the at least one amino acid modification comprises modification of an amino acid residue selected from: an aspartic acid residue, a glutamic acid residue, a histidine residue, an asparagine residue, a glutamine residue, a serine residue, a threonine residue, an alanine residue, a glycine residue, a valine residue, a leucine residue, and an isoleucine residue.
 19. The engineered clostridial toxin of claim 18, wherein the amino acid residue is substituted with a lysine residue or an arginine residue.
 20. A nucleic acid comprising a nucleic acid sequence encoding an engineered clostridial toxin according to any one of claims 1-19.
 21. A method of producing a single-chain engineered clostridial toxin protein having a light chain and a heavy chain, the method comprising expressing a nucleic acid according to claim 20 in a suitable host cell, lysing the host cell to provide a host cell homogenate containing the single-chain engineered clostridial toxin protein, and isolating the single-chain engineered clostridial toxin protein.
 22. A method of activating an engineered clostridial toxin, the method comprising providing a single-chain engineered clostridial toxin protein obtainable by the method of claim 21, contacting the polypeptide with a protease that cleaves the polypeptide at a recognition site (cleavage site) located between the light chain and heavy chain, and converting the polypeptide into a di-chain polypeptide wherein the light chain and heavy chain are joined together by a disulphide bond.
 23. An engineered clostridial toxin according to any one of claims 1-19, for use in medicine.
 24. An engineered clostridial toxin according to any one of claims 1-19, for use in the prevention or treatment of a disease or condition selected from: strabismus, blepharospasm, squint, dystonia (e.g. spasmodic dystonia, oromandibular dystonia, focal dystonia, tardive dystonia, laryngeal dystonia, limb dystonia, cervical dystonia), torticollis (e.g. spasmodic torticollis), beauty therapy (cosmetic) applications benefiting from cell/muscle incapacitation (via SNARE down-regulation or inactivation), neuromuscular disorder or condition of ocular motility (e.g. concomitant strabismus, vertical strabismus, lateral rectus palsy, nystagmus, dysthyroid myopathy), writer's cramp, blepharospasm, bruxism, Wilson's disease, tremor, tics, segmental myoclonus, spasms, spasticity due to chronic multiple sclerosis, spasticity resulting in abnormal bladder control, animus, back spasm, charley horse, tension headaches, levator pelvic syndrome, spina bifida, tardive dyskinesia, Parkinson's disease, stuttering, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, muscle pain (e.g. pain from muscle spasms), headache pain (e.g. tension headache), brow furrows, skin wrinkles, cancer, uterine disorders, uro-genital disorders, urogenital-neurological disorders, chronic neurogenic inflammation, and a smooth muscle disorder.
 25. An engineered clostridial toxin substantially as described herein and with reference to the accompanying drawings. 